Methods and assays relating to circulating tumor cells

ABSTRACT

The technology described herein relates to methods of detecting circulating tumor cells (CTCs), e.g. by detecting changes in the expression of certain CTC marker genes. Aberrant expression of CTC marker genes, e.g. changes in expression indicative of CTCs can also be targeted in order to treat cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 61/918,816 filed Dec. 20, 2013 and 61/937,883 filed Feb. 10, 2014, the contents of which are incorporated herein by reference in their entirety.

GOVERNMENT SUPPORT

This invention was made with federal funding under Grant Nos. 2R01CA129933 awarded by the National Institutes of Health. The U.S. government has certain rights in the invention.

TECHNICAL FIELD

The technology described herein relates to the diagnosis and treatment of cancer.

BACKGROUND

Circulating Tumor Cells (CTCs) are shed from primary tumors into the bloodstream, mediating the spread of cancer to distant organs (metastasis). Thus, the presence of circulating tumor cells (CTCs) in the bloodstream ultimately leads to spread of cancer to distant organs. However, CTCs are rare, estimated at one to ten tumor cells among ten billion normal blood cells in a milliliter of blood. As such, their isolation and molecular analysis has posed a significant technological challenge (Pantel et al., Nat Rev Cancer 2008 8:329-340; Yu et al., J Cell Biol 2011 192:373-382).

SUMMARY

As described herein, the inventors have identified a number of genes, the expression of which is characteristic of CTCs. In particular, the expression of these genes differentiates CTCs from primary tumor cells Accordingly, provided herein are methods and assays relating to the detection of CTCs, including diagnostic and prognostic methods and assays. Further, provided herein are treatments for cancer that target these markers of CTCs, e.g., to inhibit metastasis.

In one aspect, described herein is a method of detecting circulating tumor cells (CTCs) in a sample, the method comprising: measuring the level of a PC-CTC marker gene expression product in the sample; and determining that PC-CTCs are present if the detected level of the marker gene expression product is greater than a reference level. In some embodiments, the CTCs are pancreatic cancer CTCs. In some embodiments, the method further comprises a first step of isolating the CTCs from the sample. In some embodiments, the expression product is a nucleic acid. In some embodiments, the level of the expression product is determined using a method selected from the group consisting of RT-PCR; quantitative RT-PCR; Northern blot; microarray based expression analysis; next-generation sequencing; and RNA in situ hybridization. In some embodiments, the expression product is a polypeptide. In some embodiments, the level of the expression product is determined using a method selected from the group consisting of: Western blot; immunoprecipitation; enzyme-linked immunosorbent assay (ELISA); radioimmunological assay (RIA); sandwich assay; fluorescence in situ hybridization (FISH); immunohistological staining; radioimmunometric assay; immunofluoresence assay; mass spectroscopy; FACS; and immunoelectrophoresis assay. In some embodiments, the CTC marker gene is selected from Table 7 or Table 8. In some embodiments, the CTC marker gene is selected from the group consisting of: ABI3BP; ADAMTS5; ADAMTSL1; ANG; ARSA; C1RL; C3; C4A; C4B; CCDC80; CD109; CHI3L1; CLEC3B; CMTM3; CMTM7; COL14A1; COL1A2; COL3A1; COL4A6; CSF1; DAG1; DCN; DMKN; FBLN1; FGF1; FMOD; GPC3; GPC4; HMGB1; IFNAR2; IGFBP5; IL16; LAMA4; LTBP4; MFAP1A; NID2; OGN; PDAP1; PF4; PLAT; PODN; PRELP; RSPO1; SERPING1; SLURP1; SOD3; SPARC; SPOCK2; SPON2; SULF1; SULF2; TGFB2; TGM2; THBD; THBS1; THSD4; TIMP2; TNXB; TPT1; TWSG1 and WNT4. In some embodiments, the CTC marker gene is selected from the group consisting of: ALDH1A1; ALDH1A2; IGFBP5; KLF4; DCN; SPARC; WNT; TGFB2; VEGF; COL1A2; COL3A1; and TIMP2. In some embodiments, the CTC marker gene is selected from the group consisting of: ALDH1A2; IGFBP5; KLF4; DCN; and SPARC.

In one aspect, described herein is a method of treating cancer in a subject, the method comprising administering a therapeutically effective amount of a CTC marker gene-targeted therapy to the subject. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the CTC marker gene-targeted therapy comprises an inhibitor of a CTC marker gene. In some embodiments, the inhibitor is an antibody reagent. In some embodiments, the inhibitor is an inhibitory nucleic acid reagent. In some embodiments, the CTC marker gene-targeted therapy comprises a CTC marker gene-binding antibody reagent and a chemotherapeutic agent. In some embodiments, the subject is a subject determined to have an elevated level of CTCs and/or an elevated level of a CTC marker gene present in the blood and/or stroma of the cancer.

In one aspect, described herein is a method of determining if a subject is likely to respond to treatment with a CTC marker gene-targeted therapy, the method comprising measuring the level of a CTC marker gene expression product present in the blood and/or stroma of a cancer; and determining that the subject is likely to respond to the treatment if the level of the expression product is increased relative to a reference level. In some embodiments, the method further comprises a first step of isolating the CTCs from the sample. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the expression product is a nucleic acid. In some embodiments, the level of the expression product is determined using a method selected from the group consisting of RT-PCR; quantitative RT-PCR; Northern blot; microarray based expression analysis; next-generation sequencing; and RNA in situ hybridization. In some embodiments, the expression product is a polypeptide. In some embodiments, the level of the expression product is determined using a method selected from the group consisting of: Western blot; immunoprecipitation; enzyme-linked immunosorbent assay (ELISA); radioimmunological assay (RIA); sandwich assay; fluorescence in situ hybridization (FISH); immunohistological staining; radioimmunometric assay; immunofluoresence assay; mass spectroscopy; FACS; and immunoelectrophoresis assay. In some embodiments, the PC-CTC marker gene is selected from Table 7 or Table 8. In some embodiments, the CTC marker gene is selected from the group consisting of: ABI3BP; ADAMTS5; ADAMTSL1; ANG; ARSA; C1RL; C3; C4A; C4B; CCDC80; CD109; CHI3L1; CLEC3B; CMTM3; CMTM7; COL14A1; COL1A2; COL3A1; COL4A6; CSF1; DAG1; DCN; DMKN; FBLN1; FGF1; FMOD; GPC3; GPC4; HMGB1; IFNAR2; IGFBP5; IL16; LAMA4; LTBP4; MFAP1A; NID2; OGN; PDAP1; PF4; PLAT; PODN; PRELP; RSPO1; SERPING1; SLURP1; SOD3; SPARC; SPOCK2; SPON2; SULF1; SULF2; TGFB2; TGM2; THBD; THBS1; THSD4; TIMP2; TNXB; TPT1; TWSG1 and WNT4. In some embodiments, the CTC marker gene is selected from the group consisting of: ALDH1A1; ALDH1A2; IGFBP5; KLF4; DCN; SPARC; WNT; TGFB2; VEGF; COL1A2; COL3A1; and TIMP2. In some embodiments, the CTC marker gene is selected from the group consisting of: ALDH1A2; IGFBP5; KLF4; DCN; and SPARC.

In one aspect, described herein is a method of monitoring the treatment of a subject, the method comprising: administering a cancer therapy to a subject in need thereof; measuring the level of a CTC marker gene expression product present in the blood and/or stroma of a cancer; and determining that the subject is responding if the level of the CTC marker gene expression product is decreased relative to the reference level and determining that the subject is not responding to the treatment if the CTC marker gene expression product is not decreased relative to the reference level. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the reference level is the level of the gene expression product in the patient prior to the administering step. In some embodiments, the method further comprises a first step of isolating the CTCs from the sample. In some embodiments, the expression product is a nucleic acid. In some embodiments, the level of the expression product is determined using a method selected from the group consisting of RT-PCR; quantitative RT-PCR; Northern blot; microarray based expression analysis; next-generation sequencing; and RNA in situ hybridization. In some embodiments, the expression product is a polypeptide. In some embodiments, the level of the expression product is determined using a method selected from the group consisting of: Western blot; immunoprecipitation; enzyme-linked immunosorbent assay (ELISA); radioimmunological assay (RIA); sandwich assay; fluorescence in situ hybridization (FISH); immunohistological staining; radioimmunometric assay; immunofluoresence assay; mass spectroscopy; FACS; and immunoelectrophoresis assay. In some embodiments, the PC-CTC marker gene is selected from Table 7 or Table 8. In some embodiments, the CTC marker gene is selected from the group consisting of: ABI3BP; ADAMTS5; ADAMTSL1; ANG; ARSA; C1RL; C3; C4A; C4B; CCDC80; CD109; CHI3L1; CLEC3B; CMTM3; CMTM7; COL14A1; COL1A2; COL3A1; COL4A6; CSF1; DAG1; DCN; DMKN; FBLN1; FGF1; FMOD; GPC3; GPC4; HMGB1; IFNAR2; IGFBP5; IL16; LAMA4; LTBP4; MFAP1A; NID2; OGN; PDAP1; PF4; PLAT; PODN; PRELP; RSPO1; SERPING1; SLURP1; SOD3; SPARC; SPOCK2; SPON2; SULF1; SULF2; TGFB2; TGM2; THBD; THBS1; THSD4; TIMP2; TNXB; TPT1; TWSG1 and WNT4. In some embodiments, the CTC marker gene is selected from the group consisting of: ALDH1A1; ALDH1A2; IGFBP5; KLF4; DCN; SPARC; WNT; TGFB2; VEGF; COL1A2; COL3A1; and TIMP2. In some embodiments, the CTC marker gene is selected from the group consisting of: ALDH1A2; IGFBP5; KLF4; DCN; and SPARC.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C demonstrate the isolation and characterization of CTCs. FIG. 1A depicts a schematic of CTC-iChip negative IFD system. FIG. 1B depicts a graph of mouse WBC depletion consistency between normal and cancer mouse models. WBC depletion shown in log 10. FIG. 1C depicts a graph of CTC enumeration by immunofluorescent staining (CK+/CD45−/DAPI+) from normal and KPC mice.

FIG. 2 depicts schematics of principal component analysis of single cell samples.

FIGS. 3A-3B demonstrate that epithelial, mesenchymal, and stem cell genes are differentially expressed in CTC-c cells vs Tumors. Depicted are boxplot of genes that are A) downregulated (FIG. 3A) and upregulated (FIG. 3B) in CTC-c cells vs Tumors. Bar=median, box plot=quartiles, scale in log 10(rpm).

FIGS. 4A-4C demonstrate CTC-iChip characterization. FIG. 4A depicts a graph of the percent of WBC deflected (y-axis) as a function of the number of anti-CD45 beads per WBC (x-axis). FIG. 4B depicts a graph of the recovery of mouse PDAC cell line NB508 spiked into normal mouse blood (4 independent experiments shown). FIG. 4C depicts a graph of the captured CTCs/mL of blood from syngeneic orthotopic PDAC tumors using NB508 cell line.

FIG. 5A depicts a table of KPC mouse genotype and characteristics. FIG. 5B depicts graphs of quality metrics of single cell sequencing with % of reads aligned and total unique alignments for cell lines (NB508, MEF), CTCs, WBC, and diluted bulk RNA from matched primary tumors. FIG. 5C depicts graphs of single cell heterogeneity using mean intra-cluster correlation coefficient for each cluster (rights) and between single cell primary tumor (TuGMP3), cancer cell line (NB508), and all CTCs (Cluster 1, 3, 4, 5, 9). Circle=mean, Range=95% CI.

FIG. 6 depicts boxplot graphs of ECM protein gene enriched in CTC-c compared to bulk primary tumors and single cell primary tumors. Bar=median, boxplot—quartiles, scale in log 10 (rpm).

FIG. 7 depicts a heatmap expression profile of human pancreatic CTCs from 3 patients. Epithelial genes used to define CTCs and enriched extracellular proteins shown. Expression shown in log 10 scale.

FIG. 8 depicts a graph of quantitative RT-PCR of SPARC expression in human pancreatic cancer cell lines.

FIG. 9 depicts invasion assays. Decreases in invasion through Matrigel of PDAC2 and PDAC 3 cell lines with shRNA against SPARC (ShF1 and ShF3) were observed. shNT=Non-target shRNA

FIG. 10 depicts a graph of the number of mice with detectable metastases by in vivo luciferase imaging in non-target shRNA (NT) and SPARC shRNA (SHF1).

FIG. 11 depicts a schematic of the process of determining CTC heterogeneity.

FIGS. 12A-12C demonstrate that CTC-Enriched Genes are Found in Epithelial and Stromal Components of Primary Tumors. Depicted are expression boxplots of (FIG. 12A) Aldh1a2 stem cell and CTC highly enriched genes (FIG. 12B) Klf4 and (FIG. 12C) Igfbp5 genes. Bar=median, box plot=quartiles, scale in log 10(rpm).

FIG. 13 demonstrates that human and mouse CTCs across different epithelial cancer express high levels of ECM protein genes. Depicted are expression boxplot of highly expressed ecm genes in human pdac, breast (br), and prostate (pr) ctcs. bar, median; boxplot, quartiles; scale in log 10(rpm). holm-adjusted p value<0.05 (*), 0.01 (**), 0.001 (***).

FIGS. 14A-14E demonstrate that SPARC expression in human PDAC enhances invasion and metastasis. FIG. 14A depicts a graph of proliferation of PDAC3 cell lines determined by MTT. FIG. 14B depicts a graph of tumor spheres in PDAC3 shNT versus shSPARC counted per 43 field (error bars represent SD). FIG. 14C depicts a graph of invasion of shSPARC and shNT cell lines quantitated by number of nuclei/203 field. p value<0.01 (**), 0.001 (***), 0.0001 (****). Error bars represent SD. FIG. 14D depicts a graph of Percentage of detectable lung metastases by in vivo luciferase imaging after 3 weeks after tail vein inoculation of PDAC3 cell lines. Fisher's exact test p value is shown. FIG. 14E depicts a graph of normalized metastasis burden in mice with orthotopic pancreatic tumors from PDAC3 cell lines. Error bars represent SD (*p<0.05).

FIG. 15 depicts a Summary Model of the Role of Pancreatic CTCs in the Metastatic Cascade. Shown are the heterogeneous subsets of pancreatic CTCs with a focus on the most prominent classical CTC group, which are enriched for coexpression of epithelial (keratin) and stromal (Sparc) genes.

FIG. 16A depicts a graph of PDAC2 shRNA cell lines by qRT-PCR. Average shown with max and min RQ (error bars). FIG. 16B depicts a graph of proliferation rates by MTT assay similar in PDAC2 cell line between shNT and shSPARC stable lines. FIG. 16C depicts a graph of tumor sphere invasion assay (error bars=STD) formation at 2 weeks similar between shNT and shSPARC cell lines. Quantiation done per 4× magnification field (Error bars=SD). Migratory behavior reduced by shSPARC_1 & 3 as determined by (FIG. 16D) invasion assay at 48 hours.

DETAILED DESCRIPTION

As described herein, the inventors have discovered that circulating tumor cells (CTCs) are characterized by the expression of certain genes, i.e. CTC marker genes. The discovery of these CTC marker genes permit methods and assays for the detection and/or measurement of CTC levels, e.g. CTC levels in a sample from a subject. These methods and assays can provide improved speed and accuracy in the measurement of CTC levels. Furthermore, because the expression of these marker genes distinguishes CTCs from other cells, e.g., other circulating cells and/or normal tumor cells, therapies can be targeted against CTCs by binding to and/or inhibiting these marker gene expression products to reduce the level and/or metastatic potential of CTCs.

As used herein, “circulating tumor cell” or “CTC” refers to tumor cells which are shed from a tumor and present in the blood, i.e. in circulation. Cell markers (e.g. marker genes) that can be used to identify and/or isolate CTCs from other components of the blood are described below herein. In some embodiments, a CTC can be a pancreatic cancer CTC.

In one aspect, described herein is a method of detecting circulating tumor cells (CTCs) in a sample, the method comprising measuring the level of a CTC marker gene expression product in the sample; and determining that CTCs are present if the detected level of the marker gene expression product is greater than a reference level.

As described herein, the inventors have discovered that a number of genes are differentially regulated in CTCs, e.g. as compared to non-circulating tumor cells. Accordingly, there are provided herein methods and assays relating to the measurement of CTC levels. Elevated CTC levels can indicate a poor prognosis, e.g. an increased risk of metastatsis. Accordingly, provided herein are methods and assays related to the prognosis, risk assessment, and treatment of subjects having cancer. In certain embodiments, the assays and methods are directed to determination and/or measurement of the expression level of a gene product (e.g. protein and/or gene transcript such as mRNA) in a biological sample of a subject. In certain embodiments the assays and methods are directed to determination of the expression level of a gene product of at least two genes in a biological sample of a subject, i.e. at least two genes, at least three genes, at least four genes, at least five genes, at least six genes, at least seven genes, at least eight genes, at least nine genes, at least 10 genes . . . at least 15 genes, . . . at least 25 genes, . . . at least 30 genes, or more genes, or any number of genes selected from Table 7, Table 8, and/or Table 14 as described herein.

In some embodiments, the marker gene(s) is selected from the group consisting of ABI3BP; ADAMTS5; ADAMTSL1; ANG; ARSA; C1RL; C3; C4A; C4B; CCDC80; CD109; CHI3L1; CLEC3B; CMTM3; CMTM7; COL14A1; COL1A2; COL3A1; COL4A6; CSF1; DAG1; DCN; DMKN; FBLN1; FGF1; FMOD; GPC3; GPC4; HMGB1; IFNAR2; IGFBP5; IL16; LAMA4; LTBP4; MFAP1A; NID2; OGN; PDAP1; PF4; PLAT; PODN; PRELP; RSPO1; SERPING1; SLURP1; SOD3; SPARC; SPOCK2; SPON2; SULF1; SULF2; TGFB2; TGM2; THBD; THBS1; THSD4; TIMP2; TNXB; TPT1; TWSG1 and WNT4. In some embodiments, the assays, methods, and systems described herein are directed to determination of the expression level of a gene product of at least two genes in a biological sample of a subject, e.g. at least two genes, or at least three genes, or at least four genes, or, e.g. all of the following genes: ABI3BP; ADAMTS5; ADAMTSL1; ANG; ARSA; C1RL; C3; C4A; C4B; CCDC80; CD109; CHI3L1; CLEC3B; CMTM3; CMTM7; COL14A1; COL1A2; COL3A1; COL4A6; CSF1; DAG1; DCN; DMKN; FBLN1; FGF1; FMOD; GPC3; GPC4; HMGB1; IFNAR2; IGFBP5; IL16; LAMA4; LTBP4; MFAP1A; NID2; OGN; PDAP1; PF4; PLAT; PODN; PRELP; RSPO1; SERPING1; SLURP1; SOD3; SPARC; SPOCK2; SPON2; SULF1; SULF2; TGFB2; TGM2; THBD; THBS1; THSD4; TIMP2; TNXB; TPT1; TWSG1 and WNT4.

In some embodiments, the marker gene(s) is selected from the group consisting of ALDH1A1; ALDH1A2; IGFBP5; KLF4; DCN; SPARC; WNT; TGFB2; VEGF; COL1A2; COL3A1; and TIMP2. In some embodiments, the assays, methods, and systems described herein are directed to determination of the expression level of a gene product of at least two genes in a biological sample of a subject, e.g. at least two genes, or at least three genes, or at least four genes, or, e.g. all of the following genes: ALDH1A1; ALDH1A2; IGFBP5; KLF4; DCN; SPARC; WNT; TGFB2; VEGF; COL1A2; COL3A1; and TIMP2.

In some embodiments, the marker gene(s) is selected from the group consisting of ALDH1A2; IGFBP5; KLF4; DCN; and SPARC. In some embodiments, the assays, methods, and systems described herein are directed to determination of the expression level of a gene product of at least two genes in a biological sample of a subject, e.g. at least two genes, or at least three genes, or at least four genes, or, e.g. all of the following genes: ALDH1A2; IGFBP5; KLF4; DCN; and SPARC.

In some embodiments, the marker gene(s) is selected from the group consisting of ALDH1A2; IGFBP5; KLF4; and DCN. In some embodiments, the assays, methods, and systems described herein are directed to determination of the expression level of a gene product of at least two genes in a biological sample of a subject, e.g. at least two genes, or at least three genes, or e.g. all of the following genes: ALDH1A2; IGFBP5; KLF4; and DCN.

In some embodiments, the marker gene(s) is selected from the group consisting of TPT1; HMGB1; SPON 2; SPARC; and ARSA. In some embodiments, the assays, methods, and systems described herein are directed to determination of the expression level of a gene product of at least two genes in a biological sample of a subject, e.g. at least two genes, or at least three genes, or at least four genes, or, e.g. all of the following genes: TPT1; HMGB1; SPON 2; SPARC; and ARSA.

In some embodiments, the marker gene(s) is selected from the group consisting of IL6ST; ARSA; TIMP2; CD55; SULF2; ITGA6; SDC4; CDON; and SV2A. In some embodiments, the assays, methods, and systems described herein are directed to determination of the expression level of a gene product of at least two genes in a biological sample of a subject, e.g. at least two genes, or at least three genes, or at least four genes, or at least five genes, or at least six genes, or at least seven genes, or at least eight genes or, e.g. all of the following genes: IL6ST; ARSA; TIMP2; CD55; SULF2; ITGA6; SDC4; CDON; and SV2A. In some embodiments, the level of polypeptide expression products are determined for the marker gene(s) is selected from the group consisting of IL6ST; ARSA; TIMP2; CD55; SULF2; ITGA6; SDC4; CDON; and SV2A, e.g. because, as described herein, RNA levels of cell surface proteins are lower than polypeptide levels.

TABLE 7 Exemplary mouse marker genes MOUSE GENE SYMBOL Gene Name Abcb1b ATP-binding cassette, sub-family B (MDR/TAP), member 1B Abi3bp ABI gene family, member 3 (NESH) binding protein Ablim3 actin binding LIM protein family, member 3 Acad9 acyl-Coenzyme A dehydrogenase family, member 9 Acbd3 acyl-Coenzyme A binding domain containing 3 Acini apoptotic chromatin condensation inducer 1 Actb actin, beta Actg1 predicted gene 8543; actin-like 8; predicted gene 7505; predicted gene 12715; predicted gene 12003; predicted gene 8399; predicted gene 6375; actin, gamma, cytoplasmic 1; similar to gamma-actin; predicted gene 4667; similar to cytoplasmic beta-actin; predicted gene 16385 Adamts5 similar to a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 5 (aggrecanase-2); a disintegrin-like and metallopeptidase (reprolysin type) with thrombospondin type 1 motif, 5 (aggrecanase-2) Adamtsl1 ADAMTS-like 1 Add3 adducin 3 (gamma) Aebp1 AE binding protein 1 Agap1 ArfGAP with GTPase domain, ankyrin repeat and PH domain 1 Akap13 A kinase (PRKA) anchor protein 13 Akap2 A kinase (PRKA) anchor protein 2; paralemmin 2 Akr1b3 aldo-keto reductase family 1, member B3 (aldose reductase) Akt2 similar to RAC-beta serine/threonine-protein kinase (RAC-PK-beta) (Protein kinase Akt-2) (Protein kinase B, beta) (PKB beta); thymoma viral proto-oncogene 2; similar to serine/threonine kinase Aldh1a1 aldehyde dehydrogenase family 1, subfamily A1 Aldh1a2 aldehyde dehydrogenase family 1, subfamily A2 Alox12 arachidonate 12-lipoxygenase Amfr autocrine motility factor receptor Amhr2 anti-Mullerian hormone type 2 receptor Ang angiogenin, ribonuclease, RNase A family, 5 Ankrd11 ankyrin repeat domain 11 Ankrd12 ankyrin repeat domain 12; similar to Ankrd12 protein Ankrd17 ankyrin repeat domain 17 Ano6 anoctamin 6 Anp32a acidic (leucine-rich) nuclear phosphoprotein 32 family, member A Anxa7 annexin A7 Ap1s3 predicted gene 8532; similar to adaptor-related protein complex AP-1, sigma 3; adaptor-related protein complex AP-1, sigma 3 Ap3s1 predicted gene 7603; adaptor-related protein complex 3, sigma 1 subunit; predicted gene 5610 Ap4e1 adaptor-related protein complex AP-4, epsilon 1 Aplp1 amyloid beta (A4) precursor-like protein 1 Apol9a apolipoprotein L 9b; apolipoprotein L 9a App amyloid beta (A4) precursor protein Aqp1 aquaporin 1 Arap2 predicted gene 336; ArfGAP with RhoGAP domain, ankyrin repeat and PH domain 2 Arf2 ADP-ribosylation factor 2 Arf3 ADP-ribosylation factor 3 Arf5 similar to ADP-ribosylation factor; ADP-ribosylation factor 5 Arhgap28 Rho GTPase activating protein 28 Arhgap29 Rho GTPase activating protein 29 Arhgap5 Rho GTPase activating protein 5 Arhgef12 predicted gene 7281; predicted gene 5831; similar to SP140 nuclear body protein (predicted); Rho guanine nucleotide exchange factor (GEF) 12 Arid1a similar to AT rich interactive domain 1A isoform a; AT rich interactive domain 1A (SWI-like) Arid4a AT rich interactive domain 4A (RBP1-like) Arid4b AT rich interactive domain 4B (RBP1-like) Arid5b similar to modulator recognition factor 2; AT rich interactive domain 5B (MRF1-like) Arl3 ADP-ribosylation factor-like 3 Arl4d ADP-ribosylation factor-like 4D; hypothetical protein LOC100044157 Arl6ip5 ADP-ribosylation factor-like 6 interacting protein 5 Armcx3 armadillo repeat containing, X-linked 3; hypothetical protein LOC100044266; predicted gene 9299 Arpc2 predicted gene 5492; actin related protein 2/3 complex, subunit 2 Arsa arylsulfatase A Arsb arylsulfatase B Ascc3 activating signal cointegrator 1 complex subunit 3 Atf3 activating transcription factor 3 Atg3 autophagy-related 3 (yeast) Atp1a1 ATPase, Na+/K+ transporting, alpha 1 polypeptide Atp1b1 ATPase, Na+/K+ transporting, beta 1 polypeptide Atp2b1 ATPase, Ca++ transporting, plasma membrane 1 Atp6v1a ATPase, H+ transporting, lysosomal V1 subunit A Atxn2 ataxin 2 B230120H23Rik RIKEN cDNA B230120H23 gene B2m beta-2 microglobulin BC003331 similar to odorant response abnormal 4; cDNA sequence BC003331 BC005537 cDNA sequence BC005537 BC005561 THO complex 2; cDNA sequence BC005561 BC013529 cDNA sequence BC013529 Baz2a bromodomain adjacent to zinc finger domain, 2A Bbs4 Bardet-Biedl syndrome 4 (human) Bbx bobby sox homolog (Drosophila) Bcam basal cell adhesion molecule Bcl10 B-cell leukemia/lymphoma 10; predicted gene 6141 Bdp1 B double prime 1, subunit of RNA polymerase III transcription initiation factor IIIB Bicc1 bicaudal C homolog 1 (Drosophila) Bicd1 bicaudal D homolog 1 (Drosophila) Birc6 baculoviral IAP repeat-containing 6 Blvrb biliverdin reductase B (flavin reductase (NADPH)) Bnc1 basonuclin 1 Bnc2 basonuclin 2 Bod1l biorientation of chromosomes in cell division 1-like Bptf bromodomain PHD finger transcription factor Braf Braf transforming gene Brd2 similar to mKIAA4005 protein; bromodomain containing 2 Brd4 bromodomain containing 4 Brp44l similar to brain protein 44-like protein; brain protein 44-like; predicted gene 3452; predicted gene 8219 Bst2 bone marrow stromal cell antigen 2 Btbd2 BTB (POZ) domain containing 2 Btbd7 BTB (POZ) domain containing 7 Btf3 predicted gene 9308; basic transcription factor 3; predicted gene 3531; predicted gene 7973 Btg2 B-cell translocation gene 2, anti-proliferative Bzw1 predicted gene 11652; predicted gene 5191; basic leucine zipper and W2 domains 1 C1d C1D nuclear receptor co-repressor C1ra complement component 1, r subcomponent; predicted gene 8551 C1rl complement component 1, r subcomponent-like C1s similar to Complement component 1, s subcomponent; complement component 1, s subcomponent C2 complement component 2 (within H-2S) C3 complement component 3; similar to complement component C3 prepropeptide, last C4a similar to Complement C4 precursor; complement component 4A (Rodgers blood group); similar to complement C4; complement component 4B (Childo blood group) C4b similar to Complement C4 precursor; complement component 4A (Rodgers blood group); similar to complement C4; complement component 4B (Childo blood group) Calm1 predicted gene 7743; calmodulin 3; calmodulin 2; calmodulin 1; predicted gene 7308 Calm2 predicted gene 7743; calmodulin 3; calmodulin 2; calmodulin 1; predicted gene 7308 Cap1 CAP, adenylate cyclase-associated protein 1 (yeast) Cast calpastatin Cav1 caveolin 1, caveolae protein Ccdc109b coiled-coil domain containing 109B Ccdc34 coiled-coil domain containing 34 Ccdc80 coiled-coil domain containing 80 Ccdc88a coiled coil domain containing 88A Ccdc90a coiled-coil domain containing 90A Ccnl1 cyclin L1 Cd109 CD109 antigen Cd200 CD200 antigen; similar to MRC OX-2 antigen homolog Cd248 CD248 antigen, endosialin Cd34 CD34 antigen Cd55 CD55 antigen Cd81 CD81 antigen Cd82 CD82 antigen Cd9 CD9 antigen Cdc42ep3 CDC42 effector protein (Rho GTPase binding) 3 Cdh11 cadherin 11 Cdh3 cadherin 3 Cdk13 cell division cycle 2-like 5 (cholinesterase-related cell division controller) Cdon cell adhesion molecule-related/down-regulated by oncogenes Celf2 CUG triplet repeat, RNA binding protein 2 Cep164 centrosomal protein 164 Cep57 centrosomal protein 57 Cfh complement component factor h; similar to complement component factor H Cfl1 cofilin 1, non-muscle; similar to Cofilin-1 (Cofilin, non-muscle isoform); predicted gene 6180 Cfl2 cofilin 2, muscle Chd1 chromodomain helicase DNA binding protein 1 Chd2 chromodomain helicase DNA binding protein 2 Chi3l1 chitinase 3-like 1 Chst4 carbohydrate (chondroitin 6/keratan) sulfotransferase 4 Cish cytokine inducible SH2-containing protein Clcn3 chloride channel 3 Cldn15 claudin 15 Cldn25 predicted gene 16492 Clec1b C-type lectin domain family 1, member b Clec3b C-type lectin domain family 3, member b Clic4 chloride intracellular channel 4 (mitochondrial) Clip1 CAP-GLY domain containing linker protein 1 Clip3 CAP-GLY domain containing linker protein 3 Cln8 ceroid-lipofuscinosis, neuronal 8 Cmah cytidine monophospho-N-acetylneuraminic acid hydroxylase Cmtm3 CKLF-like MARVEL transmembrane domain containing 3 Cmtm7 CKLF-like MARVEL transmembrane domain containing 7 Cnot6l CCR4-NOT transcription complex, subunit 6-like Cobl cordon-bleu Cobll1 Cobl-like 1 Col14a1 collagen, type XIV, alpha 1 Col1a2 collagen, type I, alpha 2 Col3a1 collagen, type III, alpha 1 Col4a6 collagen, type IV, alpha 6 Colec12 collectin sub-family member 12 Coq10b hypothetical protein LOC675736; coenzyme Q10 homolog B (S. cerevisiae); predicted gene 4899 Creb3l1 cAMP responsive element binding protein 3-like 1 Creb5 RIKEN cDNA 9430076C15 gene; cAMP responsive element binding protein 5 Crebbp CREB binding protein Creg1 cellular represser of E1A-stimulated genes 1 Crim1 cysteine rich transmembrane BMP regulator 1 (chordin like) Crls1 cardiolipin synthase 1 Cryab crystallin, alpha B Cryl1 crystallin, lambda 1 Crym crystallin, mu Csda cold shock domain protein A Csf1 colony stimulating factor 1 (macrophage) Csnk1a1 casein kinase 1, alpha 1 Csrnp1 cysteine-serine-rich nuclear protein 1 Csrp1 cysteine and glycine-rich protein 1 Cuedc1 CUE domain containing 1 Cyb5 cytochrome b-5 Cybrd1 cytochrome b reductase 1 Cyp2d22 cytochrome P450, family 2, subfamily d, polypeptide 22 Cyp2s1 cytochrome P450, family 2, subfamily s, polypeptide 1 Cyr61 cysteine rich protein 61 Dab2 disabled homolog 2 (Drosophila) Dag1 dystroglycan 1 Daglb diacylglycerol lipase, beta Dapk1 death associated protein kinase 1 Dcn decorin Ddr1 discoidin domain receptor family, member 1 Ddr2 discoidin domain receptor family, member 2 Ddx3x DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 3, X-linked Ddx5 DEAD (Asp-Glu-Ala-Asp) box polypeptide 5; predicted gene 12183 Dennd5a DENN/MADD domain containing 5A; similar to Rab6 interacting protein 1 Dhx15 DEAH (Asp-Glu-Ala-His) box polypeptide 15 Diap1 diaphanous homolog 1 (Drosophila) Dlgap4 discs, large homolog-associated protein 4 (Drosophila) Dmkn dermokine Dnaja2 DnaJ (Hsp40) homolog, subfamily A, member 2 Dnajb9 predicted gene 6568; DnaJ (Hsp40) homolog, subfamily B, member 9 Dnajc1 DnaJ (Hsp40) homolog, subfamily C, member 1 Dnmt1 DNA methyltransferase (cytosine-5) 1 Dpp4 dipeptidylpeptidase 4 Dpysl2 dihydropyrimidinase-like 2 Dpysl3 dihydropyrimidinase-like 3 Dst dystonin; hypothetical protein LOC100047109 Dtx2 deltex 2 homolog (Drosophila) Dusp1 dual specificity phosphatase 1 Dusp14 dual specificity phosphatase 14 Dusp3 dual specificity phosphatase 3 (vaccinia virus phosphatase VH1-related) Dync1i2 dynein cytoplasmic 1 intermediate chain 2 Ecd ecdysoneless homolog (Drosophila) Eea1 early endosome antigen 1 Eef1a1 predicted gene 5869; predicted gene 7161; predicted gene 7105; predicted gene 5822; similar to eukaryotic translation elongation factor 1 alpha 1; predicted gene 6192; predicted gene 6392; predicted gene 6767; predicted gene 6170; predicted gene 6548; predicted gene 6789; eukaryotic translation elongation factor 1 alpha 1 Efemp1 epidermal growth factor-containing fibulin-like extracellular matrix protein 1 Efhd2 similar to EF hand domain containing 2; EF hand domain containing 2 Efna5 ephrin A5 Egr1 early growth response 1 Ehd2 EH-domain containing 2 Eif2s3x eukaryotic translation initiation factor 2, subunit 3, structural gene X- linked; similar to translation initiation factor elF-2 gamma subunit; predicted gene 2223 Eif3a eukaryotic translation initiation factor 3, subunit A Elf1 E74-like factor 1 Elovl6 predicted gene 11295; ELOVL family member 6, elongation of long chain fatty acids (yeast) Emp2 epithelial membrane protein 2 Enpp2 ectonucleotide pyrophosphatase/phosphodiesterase 2 Enpp4 ectonucleotide pyrophosphatase/phosphodiesterase 4 Esam endothelial cell-specific adhesion molecule Esf1 ESF1, nucleolar pre-rRNA processing protein, homolog (S. cerevisiae) Espn espin Esyt3 family with sequence similarity 62 (C2 domain containing), member C Etfa predicted gene 2893; electron transferring flavoprotein, alpha polypeptide Evpl envoplakin Exoc4 exocyst complex component 4 F11r F11 receptor Faim2 Fas apoptotic inhibitory molecule 2 Fam117a family with sequence similarity 117, memberA Fam134b family with sequence similarity 134, member B Fam53b family with sequence similarity 53, member B Fam63b RIKEN cDNA B230380D07 gene Fam76a predicted gene 7527; family with sequence similarity 76, member A Fam84b RIKEN cDNA D330050I23 gene Fas Fas (TNF receptor superfamily member 6) Fbln1 fibulin 1 Fermt2 fermitin family homolog 2 (Drosophila) Fgf1 fibroblast growth factor 1 Fhl1 four and a half LIM domains 1 Filip1l filamin A interacting protein 1-like Fkbp5 FK506 binding protein 5 Flii flightless I homolog (Drosophila); similar to cytoskeletal actin-modulating protein Flnc filamin C, gamma Flrt2 fibronectin leucine rich transmembrane protein 2 Fmo2 flavin containing monooxygenase 2 Fmod fibromodulin Fndc1 fibronectin type III domain containing 1; similar to fibronectin type III domain containing 1 Fos FBJ osteosarcoma oncogene Foxn3 forkhead box N3 Frmd4b FERM domain containing 4B Fth1 ferritin heavy chain 1 Fxyd1 FXYD domain-containing ion transport regulator 1 G3bp1 Ras-GTPase-activating protein SH3-domain binding protein 1 Gabarapl1 gamma-aminobutyric acid (GABA) A receptor-associated protein-like 1 Gadd45b growth arrest and DNA-damage-inducible 45 beta Ganab alpha glucosidase 2 alpha neutral subunit Gas1 growth arrest specific 1 Gas6 growth arrest specific 6 Gata6 GATA binding protein 6 Gbp2 guanylate binding protein 2 Gbp3 guanylate binding protein 3 Gcap14 granule cell antiserum positive 14 Gcsh predicted gene 3672; similar to Glycine cleavage system H protein, mitochondrial precursor; glycine cleavage system protein H (aminomethyl carrier) Gda guanine deaminase Gem GTP binding protein (gene overexpressed in skeletal muscle) Gfm2 G elongation factor, mitochondrial 2 Gfpt2 glutamine fructose-6-phosphate transaminase 2 Gja1 gap junction protein, alpha 1 Gjb5 gap junction protein, beta 5 Gm10052 predicted gene 10052 Gm13251 predicted gene 13251; predicted gene, OTTMUSG00000010657; RIKEN cDNA 1700029I01 gene Gm3893 similar to 4933409K07Rik protein; predicted gene, 665845; predicted gene 2490; predicted gene 10601; predicted gene 2163; predicted gene 3892; RIKEN cDNA 4933409K07 gene; predicted gene 3893 Gm6548 predicted gene 5869; predicted gene 7161; predicted gene 7105; predicted gene 5822; similar to eukaryotic translation elongation factor 1 alpha 1; predicted gene 6192; predicted gene 6392; predicted gene 6767; predicted gene 6170; predicted gene 6548; predicted gene 6789; eukaryotic translation elongation factor 1 alpha 1 Gm6578 predicted gene 6578 Gm6644 predicted gene 6644 Gm9199 predicted gene 9199 Gnb2 guanine nucleotide binding protein (G protein), beta 2 Golga4 golgi autoantigen, golgin subfamily a, 4 Golgb1 golgi autoantigen, golgin subfamily b, macrogolgin 1 Gpc3 glypican 3 Gpc4 glypican 4; similar to Glypican 4 Gpcpd1 preimplantation protein 4 Gpm6a glycoprotein m6a Gpr116 G protein-coupled receptor 116 Gpr133 G protein-coupled receptor 133 Gpr64 G protein-coupled receptor 64 Gprc5b G protein-coupled receptor, family C, group 5, member B Gpx8 glutathione peroxidase 8 (putative) Gsr similar to Glutathione reductase, mitochondrial precursor (GR) (GRase); glutathione reductase Gsta3 glutathione S-transferase, alpha 3 Gstm1 similar to Glutathione S-transferase Mu 1 (GST class-mu 1) (Glutathione S- transferase GT8.7) (pmGT10) (GST 1-1); predicted gene 5562; glutathione S-transferase, mu 1 Gstm4 glutathione S-transferase, mu 4 Gucy1a3 guanylate cyclase 1, soluble, alpha 3 H2-D1 histocompatibility 2, D region; histocompatibility 2, D region locus 1 H2-K1 histocompatibility 2, K1, K region; similar to H-2K(d) antigen H2-Q6 histocompatibility 2, Q region locus 1; histocompatibility 2, Q region locus 9; similar to H-2 class I histocompatibility antigen, L-D alpha chain precursor; histocompatibility 2, Q region locus 8; histocompatibility 2, Q region locus 2; similar to MHC class Ib antigen; histocompatibility 2, Q region locus 7; histocompatibility 2, Q region locus 6; hypothetical protein LOC100044307; similar to H-2 class I histocompatibility antigen, Q7 alpha chain precursor (QA-2 antigen); RIKEN cDNA 0610037M15 gene H3f3a predicted gene 14383; predicted gene 3835; predicted gene 14384; predicted gene 12950; predicted gene, 670915; H3 histone, family 3A; predicted gene 12657; predicted gene 6132; predicted gene 10257; predicted gene 7227; H3 histone, family 3B; predicted gene 6128; similar to histone; predicted gene 1986; predicted gene 6186; hypothetical protein LOC676337; predicted gene 6421; predicted gene 2198; predicted gene 6817; predicted gene 8095; predicted gene 12271; predicted gene 13529; predicted gene 8029; predicted gene 4938; predicted gene 7100; predicted gene 9014; similar to Histone H3.4 (Embryonic); predicted gene 7179; similar to H3 histone, family 3B; predicted gene 7900; predicted gene 2099; similar to H3 histone, family 3A; predicted gene 6749; predicted gene 6485; predicted gene 4028; predicted gene 7194 Hdac3 histone deacetylase 3 Hdac5 histone deacetylase 5 Heg1 HEG homolog 1 (zebrafish) Herpud2 HERPUD family member 2 Hes1 hairy and enhancer of split 1 (Drosophila) Hexb hexosaminidase B Hist1h1c histone cluster 1, H1c Hmgb1 predicted gene 13121; predicted gene 3160; high-mobility group (nonhistone chromosomal) protein 1-like 1; predicted gene 6090; predicted gene 3851; predicted gene 8967; predicted gene 7782; predicted gene 4587; predicted gene 4689; predicted gene 3307; predicted gene 13932; predicted gene 15059; predicted gene 3565; predicted gene 15447; predicted gene 12587; predicted gene 9012; predicted gene 6115; predicted gene 9480; high mobility group box 1; predicted gene 8423; predicted gene 5853; predicted gene 8288; predicted gene 7888; predicted gene 8594; predicted gene 15387; predicted gene 5473; predicted gene 8807; similar to high mobility group box 1; similar to 2810416G20Rik protein; predicted gene 8390; predicted gene, OTTMUSG00000005439; predicted gene 5842; predicted gene 5527; predicted gene 8563; predicted gene 2710; predicted gene 12331; predicted gene 5937; predicted gene 5504; similar to high-mobility group box 1; predicted gene 10361; predicted gene 2607; predicted gene 7422; predicted gene 10075; predicted gene 12568; predicted gene 6589; predicted gene 4383; predicted gene 8031; similar to High mobility group protein 1 (HMG-1) (High mobility group protein B1) (Amphoterin) (Heparin-binding protein p30); predicted gene 7468; predicted gene 8554 Hnrnph1 heterogeneous nuclear ribonucleoprotein H1 Hnrnph2 heterogeneous nuclear ribonucleoprotein H2 Hnrnpl heterogeneous nuclear ribonucleoprotein L Hnrnpm heterogeneous nuclear ribonucleoprotein M Hnrnpr predicted gene 6159; heterogeneous nuclear ribonucleoprotein R Hook3 hook homolog 3 (Drosophila) Hoxa5 homeo box A5 Hp1bp3 heterochromatin protein 1, binding protein 3 Hsp90aa1 predicted gene 5511; heat shock protein 90, alpha (cytosolic), class A member 1 Hsp90ab1 heat shock protein 90 alpha (cytosolic), class B member 1 Hsp90b1 heat shock protein 90, beta (Grp94), member 1 Hspa12a heat shock protein 12A Hspa2 heat shock protein 2 Hspb1 heat shock protein 1 Hspb8 heat shock protein 8 Id1 inhibitor of DNA binding 1 Id2 inhibitor of DNA binding 2 Ier2 immediate early response 2 Ifi204 interferon activated gene 204 Ifi205 interferon activated gene 205 Ifi27l2a interferon, alpha-inducible protein 27 like 2A Ifi35 interferon-induced protein 35 Ifit3 interferon-induced protein with tetratricopeptide repeats 3 Ifitm3 interferon induced transmembrane protein 3 Ifnar2 interferon (alpha and beta) receptor 2 Ifngr1 interferon gamma receptor 1 Ifrd1 interferon-related developmental regulator 1 Ift74 intraflagellar transport 74 homolog (Chlamydomonas) Igf1r insulin-like growth factor I receptor Igfbp5 insulin-like growth factor binding protein 5 Igfbp6 insulin-like growth factor binding protein 6 Il16 interleukin 16 Il17re interleukin 17 receptor E Il6ra interleukin 6 receptor, alpha Il6st interleukin 6 signal transducer Ildr2 immunoglobulin-like domain containing receptor 2 Ilf3 interleukin enhancer binding factor 3 Impad1 inositol monophosphatase domain containing 1 Ints10 integrator complex subunit 10; similar to integrator complex subunit 10 Iqsec1 IQ motif and Sec7 domain 1 Irak4 interleukin-1 receptor-associated kinase 4 Irf2bp2 interferon regulatory factor 2 binding protein 2 Irf7 interferon regulatory factor 7 Irs2 insulin receptor substrate 2 Itch itchy, E3 ubiquitin protein ligase Itga6 integrin alpha 6 Itpr2 inositol 1,4,5-triphosphate receptor 2 Jmjd1c jumonji domain containing 1C Jun Jun oncogene Junb Jun-B oncogene Jund Jun proto-oncogene related gene d Jup junction plakoglobin Kank1 KN motif and ankyrin repeat domains 1 Kcnab1 potassium voltage-gated channel, shaker-related subfamily, beta member 1 Kdelr1 KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum protein retention receptor 1 Kdm5a lysine (K)-specific demethylase 5A Kdm6b KDM1 lysine (K)-specific demethylase 6B Kdr kinase insert domain protein receptor Keap1 kelch-like ECH-associated protein 1 Kif1b kinesin family member 1B Kif5b kinesin family member 5B Klf10 Kruppel-like factor 10 Klf2 Kruppel-like factor 2 (lung) Klf4 Kruppel-like factor 4 (gut) Klf6 Kruppel-like factor 6 Klf7 Kruppel-like factor 7 (ubiquitous) Klf9 Kruppel-like factor 9 Kpna1 karyopherin (importin) alpha 1 Kpna3 karyopherin (importin) alpha 3 Krcc1 lysine-rich coiled-coil 1 Krt14 keratin 14 Ktn1 kinectin 1 Lama4 laminin, alpha 4 Lamp2 lysosomal-associated membrane protein 2 Lars2 leucyl-tRNA synthetase, mitochondrial Lass2 LAG1 homolog, ceramide synthase 2 Lass4 LAG1 homolog, ceramide synthase 4 Lgals7 lectin, galactose binding, soluble 7 Limch1 LIM and calponin homology domains 1 Lims2 LIM and senescent cell antigen like domains 2 Lman1 lectin, mannose-binding, 1 Lpar2 lysophosphatidic acid receptor 2 Lrrc20 leucine rich repeat containing 20 Lrrc58 leucine rich repeat containing 58; predicted gene, OTTMUSG00000025724 Lrrc61 leucine rich repeat containing 61 Lrrn4 leucine rich repeat neuronal 4 Lrrn4cl LRRN4 C-terminal like Ltbp4 latent transforming growth factor beta binding protein 4 Luc7l3 RIKEN cDNA 3300001P08 gene Maf similar to c-Maf long form; avian musculoaponeurotic fibrosarcoma (v- maf) AS42 oncogene homolog Maged1 melanoma antigen, family D, 1 Magt1 magnesium transporter 1 Malat1 metastasis associated lung adenocarcinoma transcript 1 (non-coding RNA) Man1a mannosidase 1, alpha Manf mesencephalic astrocyte-derived neurotrophic factor Maoa monoamine oxidase A Map3k3 mitogen-activated protein kinase kinase kinase 3 Mapk1 mitogen-activated protein kinase 1 Mapkapk3 mitogen-activated protein kinase-activated protein kinase 3 Mapre2 microtubule-associated protein, RP/EB family, member 2 Marcksl1 MARCKS-like 1; predicted gene 9106 Mat2a methionine adenosyltransferase II, alpha Mat2b methionine adenosyltransferase II, beta Matr3 matrin 3; similar to Matrin 3 Med13l mediator complex subunit 13-like Med21 mediator complex subunit 21 Mef2c myocyte enhancer factor 2C Meis2 Meis homeobox 2 Mesdc1 mesoderm development candidate 1 Metap2 methionine aminopeptidase 2 Mettl2 methyltransferase like 2 Mettl7a1 methyltransferase like 7A1 Mfap1a similar to microfibrillar-associated protein 1A; microfibrillar-associated protein 1A; microfibrillar-associated protein 1B Mfhas1 malignant fibrous histiocytoma amplified sequence 1 Mgll monoglyceride lipase Mgst1 microsomal glutathione S-transferase 1 Mll1 myeloid/lymphoid or mixed-lineage leukemia 1 Mll3 myeloid/lymphoid or mixed-lineage leukemia 3 Morf4l2 predicted gene 5521; similar to mortality factor 4 like 2; mortality factor 4 like 2 Mpdz multiple PDZ domain protein Mphosph8 M-phase phosphoprotein 8 Mras muscle and microspikes RAS Mrgprf MAS-related GPR, member F Msn moesin Mtap1a microtubule-associated protein 1 A Mtdh metadherin Mtmr6 myotubularin related protein 6 Mut methylmalonyl-Coenzyme A mutase Mxd4 Max dimerization protein 4 Myh10 myosin, heavy polypeptide 10, non-muscle Myl7 myosin, light polypeptide 7, regulatory Mylip myosin regulatory light chain interacting protein Myst4 MYST histone acetyltransferase monocytic leukemia 4 Naa25 RIKEN cDNA C330023M02 gene Naga N-acetyl galactosaminidase, alpha Nckap1 NCK-associated protein 1 Ncoa1 similar to Nuclear receptor coactivator 1 (NCoA-1) (Steroid receptor coactivator 1) (SRC-1) (Nuclear receptor coactivator protein 1) (mNRC-1); nuclear receptor coactivator 1 Ncoa4 predicted gene 6768; nuclear receptor coactivator 4 Ncor1 nuclear receptor co-repressor 1 Ndn necdin Ndst1 N-deacetylase/N-sulfotransferase (heparan glucosaminyl) 1 Ndufa4 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4 Nedd4 neural precursor cell expressed, developmentally down-regulated 4 Nf1 neurofibromatosis 1 Nfe2l1 nuclear factor, erythroid derived 2, -like 1 Nfia nuclear factor I/A Nfic nuclear factor I/C Nfix nuclear factor I/X Nfkb2 nuclear factor of kappa light polypeptide gene enhancer in B-cells 2, p49/p100 Nfkbia nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha Nfkbiz nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, zeta Nfyc nuclear transcription factor-Y gamma Nid2 nidogen 2 Ninl ninein-like Nipal3 NIPA-like domain containing 3; similar to NIPA-like domain containing 3 Nipbl Nipped-B homolog (Drosophila) Nkain4 Na+/K+ transporting ATPase interacting 4 Nkd1 naked cuticle 1 homolog (Drosophila); similar to naked cuticle 1 homolog Nnmt nicotinamide N-methyltransferase Nod1 nucleotide-binding oligomerization domain containing 1 Npr1 natriuretic peptide receptor 1 Nr1d1 nuclear receptor subfamily 1, group D, member 1 Nr3c1 nuclear receptor subfamily 3, group C, member 1 Nr4a1 nuclear receptor subfamily 4, group A, member 1 Nrgn neurogranin Nucks1 nuclear casein kinase and cyclin-dependent kinase substrate 1 Oasl2 2′-5′ oligoadenylate synthetase-like 2 Oat ornithine aminotransferase Ogdh oxoglutarate dehydrogenase (lipoamide) Ogn osteoglycin Olfr1033 olfactory receptor 1033 Olfr613 olfactory receptor 614; hypothetical protein LOC100044261; olfactory receptor 613 Opa3 optic atrophy 3 (human) Orai3 ORAI calcium release-activated calcium modulator 3 Osr1 odd-skipped related 1 (Drosophila) Oxct1 3-oxoacid CoA transferase 1 Oxnad1 oxidoreductase NAD-binding domain containing 1 Pard3b par-3 partitioning defective 3 homolog B (C. elegans) Parp14 poly (ADP-ribose) polymerase family, member 14 Parp4 poly (ADP-ribose) polymerase family, member 4 Parvb parvin, beta; similar to parvin, beta Pbx1 pre B-cell leukemia transcription factor 1; region containing RIKEN cDNA 2310056B04gene; pre B-cell leukemia transcription factor 1 Pcdh15 protocadherin 15 Pcdhgb5 protocadherin gamma subfamily B, 5 Pcm1 pericentriolar material 1 Pdap1 PDGFA associated protein 1 Pdcd6ip programmed cell death 6 interacting protein Pde4dip phosphodiesterase 4D interacting protein (myomegalin) Pdia3 protein disulfide isomerase associated 3 Pdia4 protein disulfide isomerase associated 4 Pdpn podoplanin Pef1 penta-EF hand domain containing 1 Peli1 pellino 1 Perl period homolog 1 (Drosophila) Pf4 platelet factor 4 Pfn1 profilin 1 Pgcp plasma glutamate carboxypeptidase Pgrmc1 progesterone receptor membrane component 1 Phf21a PHD finger protein 21A Phf3 PHD finger protein 3 Phip pleckstrin homology domain interacting protein Pigt phosphatidylinositol glycan anchor biosynthesis, class T; similar to GPI transamidase component PIG-T precursor (Phosphatidylinositol-glycan biosynthesis class T protein) (Neuronal development-associated protein 7) Pik3c2a phosphatidylinositol 3-kinase, C2 domain containing, alpha polypeptide Pim1 proviral integration site 1 Pitpnm2 phosphatidylinositol transfer protein, membrane-associated 2 Pkhd1l1 polycystic kidney and hepatic disease 1-like 1 Pknox1 Pbx/knotted 1 homeobox Pla2g4a phospholipase A2, group IVA (cytosolic, calcium-dependent) Plat plasminogen activator, tissue Plce1 phospholipase C, epsilon 1 Plk1s1 non-protein coding RNA 153 Plk2 polo-like kinase 2 (Drosophila) Plod2 procollagen lysine, 2-oxoglutarate 5-dioxygenase 2 Plxdc1 plexin domain containing 1 Plxdc2 plexin domain containing 2 Plxna4 plexin A4 Pmp22 peripheral myelin protein 22 Pnrc1 proline-rich nuclear receptor coactivator 1 Podn podocan Ppap2a phosphatidic acid phosphatase type 2A Ppbp pro-platelet basic protein Ppfibp2 protein tyrosine phosphatase, receptor-type, F interacting protein, binding protein 2 Ppig peptidyl-prolyl isomerase G (cyclophilin G) Ppl periplakin Ppp1cb protein phosphatase 1, catalytic subunit, beta isoform Ppp1r12a protein phosphatase 1, regulatory (inhibitor) subunit 12A Ppp1r15a protein phosphatase 1, regulatory (inhibitor) subunit 15A; myeloid differentiation primary response gene 116 Ppp3ca protein phosphatase 3, catalytic subunit, alpha isoform Pppde1 PPPDE peptidase domain containing 1 Pqlc3 PQ loop repeat containing Prelp proline arginine-rich end leucine-rich repeat Prg4 proteoglycan 4 (megakaryocyte stimulating factor, articular superficial zone protein) Prkar2a protein kinase, cAMP dependent regulatory, type II alpha Prpf40a PRP40 pre-mRNA processing factor 40 homolog A (yeast) Prr13 proline rich 13 Prss23 protease, serine, 23 Psd pleckstrin and Sec7 domain containing Psip1 PC4 and SFRS1 interacting protein 1 Psmb2 proteasome (prosome, macropain) subunit, beta type 2 Psmd11 predicted gene 14048; proteasome (prosome, macropain) 26S subunit, non-ATPase, 11 Psmd7 proteasome (prosome, macropain) 26S subunit, non-ATPase, 7 Ptges3 predicted gene 9769; prostaglandin E synthase 3 (cytosolic); similar to Sid3177p; predicted gene 11893 Ptgis prostaglandin I2 (prostacyclin) synthase Ptgs1 prostaglandin-endoperoxide synthase 1 Ptma predicted gene 12504; predicted gene 9800; predicted gene 4617; predicted gene 6625; predicted gene 7614; similar to prothymosin alpha; prothymosin alpha; predicted gene 9009 Ptp4a2 predicted gene 13422; protein tyrosine phosphatase 4a2 Ptplad2 protein tyrosine phosphatase-like A domain containing 2 Ptprd protein tyrosine phosphatase, receptor type, D Ptprf protein tyrosine phosphatase, receptor type, F Ptrf polymerase 1 and transcript release factor Qrich1 glutamine-rich 1 Qser1 glutamine and serine rich 1 R74862 expressed sequence R74862 Rab11fip1 RAB11 family interacting protein 1 (class I) Rab1b RAB1B, member RAS oncogene family Rab5c RAB5C, member RAS oncogene family Rab6b RAB6B, member RAS oncogene family Rab7 RAB7, member RAS oncogene family Rabgap1l RAB GTPase activating protein 1-like Ralbp1 ralA binding protein 1 Raly RIKEN cDNA C130057N11 gene; hnRNP-associated with lethal yellow Rarres2 retinoic acid receptor responder (tazarotene induced) 2 Rb1cc1 RB1-inducible coiled-coil 1 Rbbp6 retinoblastoma binding protein 6 Rbbp8 retinoblastoma binding protein 8 Rbm25 RNA binding motif protein 25 Rbm27 RNA binding motif protein 27 Rbm3 predicted gene 15453; RNA binding motif protein 3 Rbpms RNA binding protein gene with multiple splicing Rdx radixin Rest RE1-silencing transcription factor Rgma RGM domain family, member A Rgs10 regulator of G-protein signalling 10 Rhob ras homolog gene family, member B Rhoj ras homolog gene family, member J Rhou ras homolog gene family, member U Rnase4 ribonuclease, RNase A family 4 Rnd3 Rho family GTPase 3 Rnf167 ring finger protein 167 Rnf20 ring finger protein 20 Rock1 Rho-associated coiled-coil containing protein kinase 1 Rock2 Rho-associated coiled-coil containing protein kinase 2 Rpp25 ribonuclease P 25 subunit (human) Rras2 related RAS viral (r-ras) oncogene homolog 2 Rspo1 R-spondin homolog (Xenopus laevis) Rtf1 Rtf1, Paf1/RNA polymerase II complex component, homolog (S. cerevisiae) Rtn1 reticulon 1 Ryk receptor-like tyrosine kinase Sarnp predicted gene 6563; SAP domain containing ribonucleoprotein Sat1 similar to spermidine/spermine N1-acetyltransferase; predicted gene 5552; spermidine/spermine N1-acetyl transferase 1 Sbsn suprabasin Scd1 stearoyl-Coenzyme A desaturase 1 Sdc4 syndecan 4 Sdpr serum deprivation response Sec62 SEC62 homolog (S. cerevisiae) Secisbp2 SECIS binding protein 2 Sema5a sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5A Senp6 similar to Sentrin-specific protease 6 (Sentrin/SUMO-specific protease SENP6) (SUMO-1-specific protease 1); SUMO/sentrin specific peptidase 6 Sep15 selenoprotein Sept9 septin 9 Serinc5 serine incorporator 5 Serpinb6b serine (or cysteine) peptidase inhibitor, clade B, member 6b Serping1 serine (or cysteine) peptidase inhibitor, clade G, member 1 Serpinh1 serine (or cysteine) peptidase inhibitor, clade H, member 1 Sesn1 sestrin 1 Setd2 SET domain containing 2 Sf3b1 splicing factor 3b, subunit 1 Sf3b4 predicted gene 7935; splicing factor 3b, subunit 4 Sfrs18 splicing factor, arginine/serine-rich 18 Shc1 predicted gene 5500; src homology 2 domain-containing transforming protein C1 Shfm1 split hand/foot malformation (ectrodactyly) type 1 Siae sialic acid acetylesterase Siah1a seven in absentia 1A Sirt2 sirtuin 2 (silent mating type information regulation 2, homolog) 2 (S. cerevisiae) Slc10a3 solute carrier family 10 (sodium/bile acid cotransporter family), member 3 Slc16a1 solute carrier family 16 (monocarboxylic acid transporters), member 1 Slc1a5 solute carrier family 1 (neutral amino acid transporter), member 5 Slc26a3 solute carrier family 26, member 3 Slc27a3 solute carrier family 27 (fatty acid transporter), member 3 Slc38a1 solute carrier family 38, member 1 Slc39a8 solute carrier family 39 (metal ion transporter), member 8 Slc43a3 solute carrier family 43, member 3 Slc4a4 solute carrier family 4 (anion exchanger), member 4 Slc6a4 solute carrier family 6 (neurotransmitter transporter, serotonin), member 4 Slc6a6 solute carrier family 6 (neurotransmitter transporter, taurine), member 6 Slc8a1 solute carrier family 8 (sodium/calcium exchanger), member 1 Slc9a3r1 solute carrier family 9 (sodium/hydrogen exchanger), member 3 regulator 1 Slpi secretory leukocyte peptidase inhibitor Sltm SAFB-like, transcription modulator Slu7 SLU7 splicing factor homolog (S. cerevisiae) Slurp1 secreted Ly6/Plaur domain containing 1 Smad4 similar to MAD homolog 4 (Drosophila); MAD homolog 4 (Drosophila) Smarca2 SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 2 Smarca5 predicted gene 13034; SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 5 Smc2 structural maintenance of chromosomes 2 Smc3 predicted gene 8892; structural maintenace of chromosomes 3 Smc4 structural maintenance of chromosomes 4 Smc6 structural maintenance of chromosomes 6 Smchd1 SMC hinge domain containing 1 Smpd3 sphingomyelin phosphodiesterase 3, neutral Snrnp70 small nuclear ribonucleoprotein 70 (U1) Sntb2 similar to beta-2-syntrophin; syntrophin, basic 2 Soat1 sterol O-acyltransferase 1 Socs3 suppressor of cytokine signaling 3 Sod3 superoxide dismutase 3, extracellular Sorbs1 sorbin and SH3 domain containing 1 Sorbs3 sorbin and SH3 domain containing 3 Sox6 SRY-box containing gene 6 Sp100 nuclear antigen Sp100 Spag9 sperm associated antigen 9 Spare secreted acidic cysteine rich glycoprotein; similar to Secreted acidic cysteine rich glycoprotein Spen SPEN homolog, transcriptional regulator (Drosophila) Spint2 serine protease inhibitor, Kunitz type 2 Spnb2 spectrin beta 2 Spock2 sparc/osteonectin, cwcv and kazal-like domains proteoglycan 2 Spon2 spondin 2, extracellular matrix protein Spop speckle-type POZ protein Src Rous sarcoma oncogene Srrm1 serine/arginine repetitive matrix 1 Ssh2 slingshot homolog 2 (Drosophila) Ssr3 signal sequence receptor, gamma St3gal1 ST3 beta-galactoside alpha-2,3-sialyltransferase 1 Stag1 stromal antigen 1 Star steroidogenic acute regulatory protein Stard5 StAR-related lipid transfer (START) domain containing 5 Stat3 similar to Stat3B; signal transducer and activator of transcription 3 Stim1 similar to Stromal interaction molecule 1; stromal interaction molecule 1 Stk10 serine/threonine kinase 10 Stk40 serine/threonine kinase 40 Stmn2 stathmin-like 2 Stra6 stimulated by retinoic acid gene 6 Strn3 striatin, calmodulin binding protein 3 Sulf1 sulfatase 1 Sulf2 sulfatase 2 Supt16h suppressor of Ty 16 homolog (S. cerevisiae) Sv2a synaptic vesicle glycoprotein 2 a Syne1 synaptic nuclear envelope 1 Syne2 synaptic nuclear envelope 2 Syt11 synaptotagmin XI; similar to synaptotagmin XI Sytl1 synaptotagmin-like 1; similar to synaptotagmin-like 1 Taf3 TAF3 RNA polymerase II, TATA box binding protein (TBP)-associated factor Taf7 TAF7 RNA polymerase II, TATA box binding protein (TBP)-associated factor Tapbp TAP binding protein Tbc1d15 TBC1 domain family, member 15 Tbcel tubulin folding cofactor E-like Tbl1x transducin (beta)-like 1 X-linked Tbx18 T-box18 Tceal8 transcription elongation factor A (SII)-like 8; similar to transcription elongation factor A (SII)-like 8 Tcf7l1 transcription factor 3 Tfdp2 transcription factor Dp 2 Tgfb1i1 transforming growth factor beta 1 induced transcript 1 Tgfb2 transforming growth factor, beta 2 Tgfbr2 transforming growth factor, beta receptor II Tgm2 transglutaminase 2, C polypeptide Thbd thrombomodulin Thbs1 thrombospondin 1; similar to thrombospondin 1 Thoc2 THO complex 2; cDNA sequence BC005561 Thrap3 thyroid hormone receptor associated protein 3; predicted gene 5898 Thsd4 thrombospondin, type I, domain containing 4 Timp2 tissue inhibitor of metalloproteinase 2 Tirap toll-interleukin 1 receptor (TIR) domain-containing adaptor protein Tlr2 toll-like receptor 2 Tm4sf1 transmembrane 4 superfamily member 1 Tm4sf5 transmembrane 4 superfamily member 5 Tmcc3 transmembrane and coiled coil domains 3 Tmco1 transmembrane and coiled-coil domains 1 Tmco7 transmembrane and coiled-coil domains 7 Tmed2 transmembrane emp24 domain trafficking protein 2; predicted gene 10698; predicted gene 7318 Tmem119 transmembrane protein 119 Tmem140 transmembrane protein 140 Tmem151a transmembrane protein 151A Tmem221 transmembrane protein 221 Tmem50a transmembrane protein 50A Tmem98 transmembrane protein 98 Tmod3 tropomodulin 3 Tmpo thymopoietin Tmsb4x thymosin, beta 4, X chromosome; similar to thymosin beta-4 Tnxb tenascin XB Tob2 transducer of ERBB2, 2 Topors topoisomerase 1 binding, arginine/serine-rich Tpm3 predicted gene 7848; predicted gene 7839; predicted gene 4157; similar to tropomyosin 3, gamma; tropomyosin 3, gamma; predicted gene 4903 Tppp3 tubulin polymerization-promoting protein family member 3 Tpt1 predicted gene 1974; tumor protein, translationally-controlled 1 pseudogene; tumor protein, translationally-controlled 1; predicted gene 14456 Trafd1 TRAF type zinc finger domain containing 1 Trib1 tribbles homolog 1 (Drosophila) Trim8 tripartite motif protein 8 Trpm7 transient receptor potential cation channel, subfamily M, member 7 Tsc22d3 TSC22 domain family, member 3 Tshz1 teashirt zinc finger family member 1 Tsix X (inactive)-specific transcript, antisense Tspan31 tetraspanin 31 Tspan5 tetraspanin 5 Ttc28 tetratricopeptide repeat domain 28 Ttc38 tetratricopeptide repeat domain 38 Tuba1a predicted gene 7172; similar to tubulin, alpha 1; tubulin, alpha 1A Tubb2a tubulin, beta 2A Twsg1 twisted gastrulation homolog 1 (Drosophila) Txndc5 thioredoxin domain containing 5 Txnrd1 thioredoxin reductase 1 Uap1 UDP-N-acetylglucosamine pyrophosphorylase 1 Uba7 ubiquitin-activating enzyme E1-like; RIKEN cDNA D330022A01 gene Ube2d1 ubiquitin-conjugating enzyme E2D 1, UBC4/5 homolog (yeast) Ube2l6 ubiquitin-conjugating enzyme E2L 6 Ube2n ubiquitin-conjugating enzyme E2N; similar to ubiquitin-conjugating enzyme E2 UbcH-ben; similar to ubiquitin-conjugating enzyme E2N; predicted gene 5943 Ube2v1 ubiquitin-conjugating enzyme E2 variant 1; predicted gene 7181; predicted gene 12502; similar to ubiquitin-conjugating enzyme E2 variant 1 Ubqln2 ubiquilin 2 Ubxn2a UBX domain protein 2A; predicted gene 6245 Ubxn4 UBX domain protein 4 Ugdh UDP-glucose dehydrogenase Upk1b uroplakin 1B Upk3b uroplakin 3B Usp16 ubiquitin specific peptidase 16 Usp2 ubiquitin specific peptidase 2 Usp25 ubiquitin specific peptidase 25 Usp54 ubiquitin specific peptidase 54 Usp8 ubiquitin specific peptidase 8 Utp20 UTP20, small subunit (SSU) processome component, homolog (yeast) Vat1 vesicle amine transport protein 1 homolog (T californica) Vim vimentin Vps13a vacuolar protein sorting 13A (yeast) Vwa5a von Willebrand factor A domain containing 5A Wac similar to WW domain-containing adapter protein with coiled-coil; WW domain containing adaptor with coiled-coil Wasf2 WAS protein family, member 2 Wdr26 WD repeat domain 26; similar to myocardial ischemic preconditioning upregulated protein 2 Wdr92 WD repeat domain 92 Wfdc1 WAP four-disulfide core domain 1 Wls G protein-coupled receptor 177 Wnt4 wingless-related MMTV integration site 4 Wrnip1 Werner helicase interacting protein 1 Wt1 similar to Wilms tumor homolog; Wilms tumor 1 homolog Wwc2 WW, C2 and coiled-coil domain containing 2 Xdh xanthine dehydrogenase Xist inactive X specific transcripts Yipf5 Yip1 domain family, member 5; predicted gene 5738 Ywhaz tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide; predicted gene 4202 Zbed6 similar to Zinc finger BED domain containing protein 4 Zbtb16 zinc finger and BTB domain containing 16 Zbtb20 zinc finger and BTB domain containing 20 Zbtb4 zinc finger and BTB domain containing 4 Zbtb7c zinc finger and BTB domain containing 7C Zc3h13 zinc finger CCCH type containing 13 Zc3h18 predicted gene 5939; zinc finger CCCH-type containing 18 Zcchc11 zinc finger, CCHC domain containing 11 Zcchc3 zinc finger, CCHC domain containing 3 Zfand6 zinc finger, AN1-type domain 6 Zfhx4 zinc finger homeodomain 4 Zfp148 zinc finger protein 148 Zfp277 zinc finger protein 277 Zfp281 zinc finger protein 281 Zfp318 zinc finger protein 318 Zfp353 zinc finger protein 353 Zfp36 zinc finger protein 36 Zfp385a zinc finger protein 385A Zfp488 zinc finger protein 488 Zfp672 zinc finger protein 672 Zfp704 zinc finger protein 704 Zmat1 zinc finger, matrin type 1 Zrsr1 zinc finger (CCCH type), RNA binding motif and serine/arginine rich 1 Zzef1 zinc finger, ZZ-type with EF hand domain 1 1110002B05Rik RIKEN cDNA 1110002B05 gene 1110003E01Rik RIKEN cDNA 1110003E01 gene 1110004F10Rik predicted gene 9169; RIKEN cDNA 1110004F10 gene; similar to small acidic protein 1500003O03Rik RIKEN cDNA 1500003003 gene; similar to EF-hand Ca2+ binding protein p22 1600029D21Rik RIKEN cDNA 1600029D21 gene 1810014B01Rik RIKEN cDNA 1810014B01 gene 1810041L15Rik RIKEN cDNA 1810041L15 gene 1810074P20Rik RIKEN cDNA 1810074P20 gene 2010107G12Rik RIKEN cDNA 2010107G12 gene 2210403K04Rik hypothetical protein LOC100042498 2310030G06Rik RIKEN cDNA 2310030G06 gene 2510002D24Rik RIKEN cDNA 2510002D24 gene 2610034B18Rik RIKEN cDNA 2610034B18 gene 2610101N10Rik RIKEN cDNA 2610101N10 gene 2810474O19Rik RIKEN cDNA 2810474O19 gene 2900002K06Rik RIKEN cDNA 2900002K06 gene 3110062M04Rik RIKEN cDNA 3110062M04 gene 4930402H24Rik RIKEN cDNA 4930402H24 gene 4930523C07Rik RIKEN cDNA 4930523C07 gene 5430435G22Rik RIKEN cDNA 5430435G22 gene 6330406I15Rik RIKEN cDNA 6330406I15 gene A130040M12Rik RIKEN cDNA A130040M12 gene AI848100 expressed sequence AI848100 Gm16897 kg:uc009lxf.1 Prrc2c kg:uc007won.1 kg:uc009ogv.1 kg:uc009iln.1 kg:uc007qca.1 Atxn7l3b kg:uc008ewj.2 kg:uc008wkn.1 kg:uc007bgn.1 Ces2g kg:uc009cvm.1 kg:uc008ehr.1 Tmem234 kg:uc012hdk.1 kg:uc008ajk.1 eg:245190:chr7:m kg:uc007qse.1 kg:uc007bvx.1 Mob3c kg:uc008dzh.1 kg:uc009okn.1 kg:uc007zts.1 kg:uc008jup.1 kg:uc008tkz.1 kg:uc007zwh.1 kg:uc008znh.1 Mau2 kg:uc009mng.1 kg:uc007ded.1 kg:uc007ctp.1 kg:uc007zak.1 eg:497210:chr14:m kg:uc007vsr.1 Mir3064 kg:uc009ize.1 Kansl1 eg:320169:chr9:p kg:uc009vev.1 kg:uc009acs.1 kg:uc009tuw.1 kg:uc007pff.1 kg:uc007vnc.1 kg:uc009igb.1 kg:uc008oki.1 kg:uc008tky.1

TABLE 8 Exemplary human marker genes HUMAN GENE SYMBOL Gene Name ABI3BP ABI family, member 3 (NESH) binding protein ABLIM3 actin binding LIM protein family, member 3 ACAD9 acyl-Coenzyme A dehydrogenase family, member 9 ACBD3 acyl-Coenzyme A binding domain containing 3 ACIN1 apoptotic chromatin condensation inducer 1 ACTB actin, beta ACTG1 actin, gamma 1 ADAMTS5 ADAM metallopeptidase with thrombospondin type 1 motif, 5 ADAMTSL1 ADAMTS-like 1 ADD3 adducin 3 (gamma) AEBP1 AE binding protein 1 AGAP1 ArfGAP with GTPase domain, ankyrin repeat and PH domain 1 AKAP13 A kinase (PRKA) anchor protein 13 AKAP2 A kinase (PRKA) anchor protein 2; paralemmin 2; PALM2-AKAP2 readthrough transcript AKT2 v-akt murine thymoma viral oncogene homolog 2 ALDH1A1 aldehyde dehydrogenase 1 family, member A1 ALDH1A2 aldehyde dehydrogenase 1 family, member A2 ALOX12 arachidonate 12-lipoxygenase AMFR autocrine motility factor receptor AMHR2 anti-Mullerian hormone receptor, type II ANG angiogenin, ribonuclease, RNase A family, 5 ANKRD11 ankyrin repeat domain 11; hypothetical protein LOC100128265 ANKRD12 ankyrin repeat domain 12 ANKRD17 ankyrin repeat domain 17 ANO6 anoctamin 6 ANP32A hepatopoietin PCn127; acidic (leucine-rich) nuclear phosphoprotein 32 family, member A ANXA7 annexin A7 AP1S3 adaptor-related protein complex 1, sigma 3 subunit AP3S1 adaptor-related protein complex 3, sigma 1 subunit AP4E1 adaptor-related protein complex 4, epsilon 1 subunit APLP1 amyloid beta (A4) precursor-like protein 1 APP amyloid beta (A4) precursor protein AQP1 aquaporin 1 (Colton blood group) ARAP2 ArfGAP with RhoGAP domain, ankyrin repeat and PH domain 2 ARF3 ADP-ribosylation factor 3 ARF5 ADP-ribosylation factor 5 ARHGAP28 Rho GTPase activating protein 28 ARHGAP29 Rho GTPase activating protein 29 ARHGAP5 Rho GTPase activating protein 5 ARHGEF12 Rho guanine nucleotide exchange factor (GEF) 12 ARID1A AT rich interactive domain 1A (SWI-like) ARID4A AT rich interactive domain 4A (RBP1-like) ARID4B AT rich interactive domain 4B (RBP1-like) ARID5B AT rich interactive domain 5B (MRF1-like) ARL3 ADP-ribosylation factor-like 3 ARL4D ADP-ribosylation factor-like 4D ARL6IP5 ADP-ribosylation-like factor 6 interacting protein 5 ARMCX3 armadillo repeat containing, X-linked 3 ARPC2 actin related protein 2/3 complex, subunit 2, 34 kDa ARSA arylsulfatase A ARSB arylsulfatase B ASCC3 activating signal cointegrator 1 complex subunit 3 ATF3 activating transcription factor 3 ATG3 ATG3 autophagy related 3 homolog (S. cerevisiae) ATP1A1 ATPase, Na+/K+ transporting, alpha 1 polypeptide ATP1B1 ATPase, Na+/K+ transporting, beta 1 polypeptide ATP2B1 ATPase, Ca++ transporting, plasma membrane 1 ATP6V1A ATPase, H+ transporting, lysosomal 70 kDa, V1 subunit A ATXN2 ataxin 2 B2M beta-2-microglobulin BAZ2A bromodomain adjacent to zinc finger domain, 2A BBS4 Bardet-Biedl syndrome 4 BBX bobby sox homolog (Drosophila) BCAM basal cell adhesion molecule (Lutheran blood group) BCL10 B-cell CLL/lymphoma 10; hypothetical LOC646626 BDP1 B double prime 1, subunit of RNA polymerase III transcription initiation factor IIIB BICC1 bicaudal C homolog 1 (Drosophila) BICD1 bicaudal D homolog 1 (Drosophila) BIRC6 baculoviral IAP repeat-containing 6 BLVRB biliverdin reductase B (flavin reductase (NADPH)) BNC1 basonuclin 1 BNC2 basonuclin 2 BOD1L biorientation of chromosomes in cell division 1-like BPTF bromodomain PHD finger transcription factor BRAF v-raf murine sarcoma viral oncogene homolog B1 BRD2 bromodomain containing 2 BRD4 bromodomain containing 4 BRP44L brain protein 44-like BST2 NPC-A-7; bone marrow stromal cell antigen 2 BTBD2 BTB (POZ) domain containing 2 BTBD7 BTB (POZ) domain containing 7 BTF3 basic transcription factor 3; basic transcription factor 3, like 1 pseudogene BTG2 BTG family, member 2 BZW1 basic leucine zipper and W2 domains 1 pseudogene 1; basic leucine zipper and W2 domains 1 like 1; basic leucine zipper and W2 domains 1 C1D C1D nuclear receptor co-repressor; similar to nuclear DNA-binding protein; similar to hCG1791993 C1RL complement component 1, r subcomponent-like C1S complement component 1, s subcomponent C2 complement component 2 C3 similar to Complement C3 precursor; complement component 3; hypothetical protein LOC100133511 C4A complement component 4A (Rodgers blood group) C4B complement component 4B (Chido blood group) CALM1 calmodulin 3 (phosphorylase kinase, delta); calmodulin 2 (phosphorylase kinase, delta); calmodulin 1 (phosphorylase kinase, delta) CALM2 calmodulin 3 (phosphorylase kinase, delta); calmodulin 2 (phosphorylase kinase, delta); calmodulin 1 (phosphorylase kinase, delta) CAP1 CAP, adenylate cyclase-associated protein 1 (yeast) CAST calpastatin CAV1 caveolin 1, caveolae protein, 22 kDa CCDC109B coiled-coil domain containing 109B CCDC34 coiled-coil domain containing 34 CCDC80 coiled-coil domain containing 80 CCDC88A coiled-coil domain containing 88A CCDC90A coiled-coil domain containing 90A CCNL1 cyclin L1 CD109 CD109 molecule CD200 CD200 molecule CD248 CD248 molecule, endosialin CD34 CD34 molecule CD55 CD55 molecule, decay accelerating factor for complement (Cromer blood group) CD81 CD81 molecule CD82 CD82 molecule CD9 CD9 molecule CDC42EP3 CDC42 effector protein (Rho GTPase binding) 3 CDH11 cadherin 11, type 2, OB-cadherin (osteoblast) CDH3 cadherin 3, type 1, P-cadherin (placental) CDK13 cell division cycle 2-like 5 (cholinesterase-related cell division controller) CDON Cdon homolog (mouse) CELF2 CUG triplet repeat, RNA binding protein 2 CEP164 centrosomal protein 164 kDa CEP57 centrosomal protein 57 kDa CFH complement factor H CFL1 cofilin 1 (non-muscle) CFL2 cofilin 2 (muscle) CHD1 chromodomain helicase DNA binding protein 1 CHD2 chromodomain helicase DNA binding protein 2 CHI3L1 chitinase 3-like 1 (cartilage glycoprotein-39) CHST4 carbohydrate (N-acetylglucosamine 6-O) sulfotransferase 4 CISH cytokine inducible SH2-containing protein CLCN3 chloride channel 3 CLDN10 claudin 10 CLDN15 claudin 15 CLDN25 claudin-like CLEC1B C-type lectin domain family 1, member B CLEC3B C-type lectin domain family 3, member B CLIC4 chloride intracellular channel 4 CLIP1 CAP-GLY domain containing linker protein 1 CLIP3 CAP-GLY domain containing linker protein 3 CLN8 ceroid-lipofuscinosis, neuronal 8 (epilepsy, progressive with mental retardation) CMAH cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMP-N-acetylneuraminate monooxygenase) pseudogene CMTM3 CKLF-like MARVEL transmembrane domain containing 3 CMTM7 CKLF-like MARVEL transmembrane domain containing 7 CNOT6L CCR4-NOT transcription complex, subunit 6-like COBL cordon-bleu homolog (mouse) COBLL1 COBL-like 1 COL14A1 collagen, type XIV, alpha 1 COL1A2 collagen, type I, alpha 2 COL3A1 collagen, type III, alpha 1 COL4A6 collagen, type IV, alpha 6 COLEC12 collectin sub-family member 12 COQ10B coenzyme Q10 homolog B (S. cerevisiae) CREB3L1 cAMP responsive element binding protein 3-like 1 CREB5 cAMP responsive element binding protein 5 CREBBP CREB binding protein CREG1 cellular repressor of E1A-stimulated genes 1 CRIM1 cysteine rich transmembrane BMP regulator 1 (chordin-like) CRLS1 cardiolipin synthase 1 CRYAB crystallin, alpha B CRYL1 crystallin, lambda 1 CRYM crystallin, mu CSDA cold shock domain protein A; cold shock domain protein A pseudogene 1 CSF1 colony stimulating factor 1 (macrophage) CSNK1A1 casein kinase 1, alpha 1 CSRNP1 cysteine-serine-rich nuclear protein 1 CSRP1 cysteine and glycine-rich protein 1 CUEDC1 CUE domain containing 1 CYBRD1 cytochrome b reductase 1 CYP2S1 cytochrome P450, family 2, subfamily S, polypeptide 1 CYR61 cysteine-rich, angiogenic inducer, 61 DAB2 disabled homolog 2, mitogen-responsive phosphoprotein (Drosophila) DAG1 dystroglycan 1 (dystrophin-associated glycoprotein 1) DAGLB diacylglycerol lipase, beta DAPK1 death-associated protein kinase 1 DCN decorin DDR1 discoidin domain receptor tyrosine kinase 1 DDR2 discoidin domain receptor tyrosine kinase 2 DDX3X DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, X-linked DDX5 DEAD (Asp-Glu-Ala-Asp) box polypeptide 5 DENND5A DENN/MADD domain containing 5A DHX15 DEAH (Asp-Glu-Ala-His) box polypeptide 15 DLGAP4 discs, large (Drosophila) homolog-associated protein 4 DMKN dermokine DNAJA2 DnaJ (Hsp40) homolog, subfamily A, member 2 DNAJB9 DnaJ (Hsp40) homolog, subfamily B, member 9 DNAJC1 DnaJ (Hsp40) homolog, subfamily C, member 1 DNMT1 DNA (cytosine-5-)-methyltransferase 1 DPP4 dipeptidyl-peptidase 4 DPYSL2 dihydropyrimidinase-like 2 DPYSL3 dihydropyrimidinase-like 3 DST dystonin DTX2 deltex homolog 2 (Drosophila) DUSP1 dual specificity phosphatase 1 DUSP14 dual specificity phosphatase 14 DUSP3 dual specificity phosphatase 3 DYNC1I2 similar to dynein cytoplasmic 1 intermediate chain 2; dynein, cytoplasmic 1, intermediate chain 2 ECD ecdysoneless homolog (Drosophila) EEA1 early endosome antigen 1 EEF1A1 eukaryotic translation elongation factor 1 alpha-like 7; eukaryotic translation elongation factor 1 alpha-like 3; similar to eukaryotic translation elongation factor 1 alpha 1; eukaryotic translation elongation factor 1 alpha 1 EFEMP1 EGF-containing fibulin-like extracellular matrix protein 1 EFHD2 EF-hand domain family, member D2 EFNA5 ephrin-A5 EGR1 early growth response 1 EHD2 EH-domain containing 2 EIF3A eukaryotic translation initiation factor 3, subunit A ELF1 E74-like factor 1 (ets domain transcription factor) ELOVL6 ELOVL family member 6, elongation of long chain fatty acids (FEN1/Elo2, SUR4/Elo3-like, yeast) EMP2 epithelial membrane protein 2 ENPP2 ectonucleotide pyrophosphatase/phosphodiesterase 2 ENPP4 ectonucleotide pyrophosphatase/phosphodiesterase 4 (putative function) ESAM endothelial cell adhesion molecule ESF1 similar to ABT1-associated protein; ESF1, nucleolar pre-rRNA processing protein, homolog (S. cerevisiae) ESPN espin ESYT3 family with sequence similarity 62 (C2 domain containing), member C ETFA electron-transfer-flavoprotein, alpha polypeptide EVPL envoplakin EXOC4 exocyst complex component 4 F11R F11 receptor FAIM2 Fas apoptotic inhibitory molecule 2 FAM117A family with sequence similarity 117, member A FAM134B family with sequence similarity 134, member B FAM53B family with sequence similarity 53, member B FAM63B family with sequence similarity 63, member B FAM76A family with sequence similarity 76, member A FAM84B family with sequence similarity 84, member B FAS Fas (TNF receptor superfamily, member 6) FBLN1 fibulin 1 FERMT2 fermitin family homolog 2 (Drosophila) FGF1 fibroblast growth factor 1 (acidic) FHL1 four and a half LIM domains 1 FILIP1L filamin A interacting protein 1-like FKBP5 FK506 binding protein 5 FLII flightless I homolog (Drosophila) FLNC filamin C, gamma (actin binding protein 280) FLRT2 fibronectin leucine rich transmembrane protein 2 FMO2 flavin containing monooxygenase 2 (non-functional) FMOD fibromodulin FNDC1 fibronectin type III domain containing 1 FOS v-fos FBJ murine osteosarcoma viral oncogene homolog FOXN3 forkhead box N3 FRMD4B FERM domain containing 4B FTH1 ferritin, heavy polypeptide 1; ferritin, heavy polypeptide-like 16; similar to ferritin, heavy polypeptide 1; ferritin, heavy polypeptide-like 3 pseudogene FXYD1 FXYD domain containing ion transport regulator 1 G3BP1 GTPase activating protein (SH3 domain) binding protein 1 GABARAPL1 GABA(A) receptors associated protein like 3 (pseudogene); GABA(A) receptor-associated protein like 1 GADD45B growth arrest and DNA-damage-inducible, beta GANAB glucosidase, alpha; neutral AB GAS1 growth arrest-specific 1 GAS6 similar to growth arrest-specific 6; growth arrest-specific 6 GATA6 GATA binding protein 6 GBP2 guanylate binding protein 2, interferon-inducible GBP3 guanylate binding protein 3 GBP7 guanylate binding protein 7 GCSH similar to Glycine cleavage system H protein, mitochondrial precursor; glycine cleavage system protein H (aminomethyl carrier); similar to Glycine cleavage system H protein, mitochondrial GDA guanine deaminase GEM GTP binding protein overexpressed in skeletal muscle GFM2 G elongation factor, mitochondrial 2 GFPT2 glutamine-fructose-6-phosphate transaminase 2 GJA1 gap junction protein, alpha 1, 43 kDa GJB5 gap junction protein, beta 5, 31.1 kDa GNB2 guanine nucleotide binding protein (G protein), beta polypeptide 2 GOLGA4 golgi autoantigen, golgin subfamily a, 4 GOLGB1 golgin B1, golgi integral membrane protein GPC3 glypican 3 GPC4 glypican 4 GPCPD1 hypothetical protein KIAA1434 GPM6A glycoprotein M6A GPR116 G protein-coupled receptor 116 GPR133 G protein-coupled receptor 133 GPR64 G protein-coupled receptor 64 GPRC5B G protein-coupled receptor, family C, group 5, member B GPX8 glutathione peroxidase 8 (putative) GSR glutathione reductase GSTA3 glutathione S-transferase alpha 3 GSTM1 glutathione S-transferase mu 1 GSTM4 glutathione S-transferase mu 4 GUCY1A3 guanylate cyclase 1, soluble, alpha 3 H3F3A H3 histone, family 3B (H3.3B); H3 histone, family 3A pseudogene; H3 histone, family 3A; similar to H3 histone, family 3B; similar to histone H3.3B HDAC3 histone deacetylase 3 HDAC5 histone deacetylase 5 HEG1 HEG homolog 1 (zebrafish) HERPUD2 HERPUD family member 2 HES1 hairy and enhancer of split 1, (Drosophila) HEXB hexosaminidase B (beta polypeptide) HIST1H1C histone cluster 1, H1c HMGB1 high-mobility group box 1; high-mobility group box 1-like 10 HNRNPH1 heterogeneous nuclear ribonucleoprotein H1 (H) HNRNPH2 ribosomal protein L36a pseudogene 51; ribosomal protein L36a pseudogene 37; ribosomal protein L36a pseudogene 49; heterogeneous nuclear ribonucleoprotein H2 (H′); ribosomal protein L36a HNRNPL similar to heterogeneous nuclear ribonucleoprotein L-like; heterogeneous nuclear ribonucleoprotein L HNRNPM heterogeneous nuclear ribonucleoprotein M HNRNPR heterogeneous nuclear ribonucleoprotein R HOOK3 hook homolog 3 (Drosophila) HOXA5 homeobox A5 HP1BP3 heterochromatin protein 1, binding protein 3 HSP90AA1 heat shock protein 90 kDa alpha (cytosolic), class A member 2; heat shock protein 90 kDa alpha (cytosolic), class A member 1 HSP90AB1 heat shock protein 90 kDa alpha (cytosolic), class B member 1 HSP90B1 heat shock protein 90 kDa beta (Grp94), member 1 HSPA12A heat shock 70 kDa protein 12A HSPA2 heat shock 70 kDa protein 2 HSPB1 heat shock 27 kDa protein-like 2 pseudogene; heat shock 27 kDa protein 1 HSPB8 heat shock 22 kDa protein 8 ID1 inhibitor of DNA binding 1, dominant negative helix-loop-helix protein ID2 inhibitor of DNA binding 2, dominant negative helix-loop-helix protein IER2 immediate early response 2 IFI35 interferon-induced protein 35 IFIT3 interferon-induced protein with tetratricopeptide repeats 3 IFITM3 interferon induced transmembrane protein 3 (1-8 U) IFNAR2 interferon (alpha, beta and omega) receptor 2 IFNGR1 interferon gamma receptor 1 IFRD1 interferon-related developmental regulator 1 IFT74 intraflagellar transport 74 homolog (Chlamydomonas) IGF1R insulin-like growth factor 1 receptor IGFBP5 insulin-like growth factor binding protein 5 IGFBP6 insulin-like growth factor binding protein 6 IL16 interleukin 16 (lymphocyte chemoattractant factor) IL17RE interleukin 17 receptor E IL6ST interleukin 6 signal transducer (gp130, oncostatin M receptor) ILDR2 immunoglobulin-like domain containing receptor 2 ILF3 interleukin enhancer binding factor 3, 90 kDa IMPAD1 inositol monophosphatase domain containing 1 INTS10 integrator complex subunit 10 IQSEC1 IQ motif and Sec7 domain 1 IRAK4 interleukin-1 receptor-associated kinase 4 IRF2BP2 interferon regulatory factor 2 binding protein 2 IRF7 interferon regulatory factor 7 IRS2 insulin receptor substrate 2 ITCH itchy E3 ubiquitin protein ligase homolog (mouse) ITGA6 integrin, alpha 6 ITPR2 inositol 1,4,5-triphosphate receptor, type 2 JMJD1C jumonji domain containing 1C JUN jun oncogene JUNB jun B proto-oncogene JUND jun D proto-oncogene JUP junction plakoglobin KANK1 KN motif and ankyrin repeat domains 1; similar to ankyrin repeat domain protein 15 isoform b KCNAB1 potassium voltage-gated channel, shaker-related subfamily, beta member 1 KDELR1 KDEL (Lys-Asp-Glu-Leu) endoplasmic reticulum protein retention receptor 1 KDM5A lysine (K)-specific demethylase 5A KDM6B lysine (K)-specific demethylase 6B KDR kinase insert domain receptor (a type III receptor tyrosine kinase) KEAP1 kelch-like ECH-associated protein 1 KIF1B kinesin family member 1B KIF5B kinesin family member 5B KLF10 Kruppel-like factor 10 KLF2 Kruppel-like factor 2 (lung) KLF4 Kruppel-like factor 4 (gut) KLF6 Kruppel-like factor 6 KLF7 Kruppel-like factor 7 (ubiquitous) KLF9 Kruppel-like factor 9 KPNA1 karyopherin alpha 1 (importin alpha 5) KPNA3 karyopherin alpha 3 (importin alpha 4) KRCC1 lysine-rich coiled-coil 1 KRT14 keratin 14 KTN1 kinectin 1 (kinesin receptor) LAMA4 laminin, alpha 4 LAMP2 lysosomal-associated membrane protein 2 LARS2 leucyl-tRNA synthetase 2, mitochondrial LASS2 LAG1 homolog, ceramide synthase 2 LASS4 LAG1 homolog, ceramide synthase 4 LGALS7 lectin, galactoside-binding, soluble, 7; lectin, galactoside-binding, soluble, 7B LIMCH1 LIM and calponin homology domains 1 LIMS2 LIM and senescent cell antigen-like domains 2 LMAN1 lectin, mannose-binding, 1 LPAR2 lysophosphatidic acid receptor 2 LRRC20 leucine rich repeat containing 20 LRRC58 leucine rich repeat containing 58 LRRC61 leucine rich repeat containing 61 LRRN4 leucine rich repeat neuronal 4 LRRN4CL LRRN4C-terminal like LTBP4 latent transforming growth factor beta binding protein 4 LUC7L3 cisplatin resistance-associated overexpressed protein MAF v-maf musculoaponeurotic fibrosarcoma oncogene homolog (avian) MAGED1 melanoma antigen family D, 1 MAGT1 magnesium transporter 1 MALAT1 metastasis associated lung adenocarcinoma transcript 1 (non-protein coding) MANF mesencephalic astrocyte-derived neurotrophic factor MAOA monoamine oxidase A MAP3K3 mitogen-activated protein kinase kinase kinase 3 MAPK1 mitogen-activated protein kinase 1 MAPKAPK3 mitogen-activated protein kinase-activated protein kinase 3 MAPRE2 microtubule-associated protein, RP/EB family, member 2 MARCKSL1 MARCKS-like 1 MAT2A methionine adenosyltransferase II, alpha MAT2B methionine adenosyltransferase II, beta MATR3 matrin 3 MED13L mediator complex subunit 13-like MED21 mediator complex subunit 21 MEF2C myocyte enhancer factor 2C MEIS2 Meis homeobox 2 MESDC1 mesoderm development candidate 1 METAP2 methionyl aminopeptidase 2 MFHAS1 malignant fibrous histiocytoma amplified sequence 1 MGLL monoglyceride lipase MGST1 microsomal glutathione S-transferase 1 MLL3 myeloid/lymphoid or mixed-lineage leukemia 3 MORF4L2 mortality factor 4 like 2 MPDZ multiple PDZ domain protein MPHOSPH8 M-phase phosphoprotein 8 MRAS muscle RAS oncogene homolog MRGPRF MAS-related GPR, member F MSN moesin MTDH metadherin MTMR6 myotubularin related protein 6 MUT methylmalonyl Coenzyme A mutase MXD4 MAX dimerization protein 4 MYH10 myosin, heavy chain 10, non-muscle MYL12A myosin, light chain 12A, regulatory, non-sarcomeric MYL7 myosin, light chain 7, regulatory MYLIP myosin regulatory light chain interacting protein MYST4 MYST histone acetyltransferase (monocytic leukemia) 4 NAA25 chromosome 12 open reading frame 30 NAGA N-acetylgalactosaminidase, alpha- NCKAP1 NCK-associated protein 1 NCOA1 nuclear receptor coactivator 1 NCOA4 nuclear receptor coactivator 4 NCOR1 nuclear receptor co-repressor 1 NDN necdin homolog (mouse) NDST1 N-deacetylase/N-sulfotransferase (heparan glucosaminyl) 1 NDUFA4 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4, 9 kDa NEDD4 neural precursor cell expressed, developmentally down-regulated 4 NF1 neurofibromin 1 NFE2L1 nuclear factor (erythroid-derived 2)-like 1 NFIA nuclear factor I/A NFIC nuclear factor I/C (CCAAT-binding transcription factor) NFIX nuclear factor I/X (CCAAT-binding transcription factor) NFKB2 nuclear factor of kappa light polypeptide gene enhancer in B-cells 2 (p49/p100) NFKBIA nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha NFKBIZ nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, zeta NFYC nuclear transcription factor Y, gamma NID2 nidogen 2 (osteonidogen) NINL ninein-like NIPAL3 NIPA-like domain containing 3 NIPBL Nipped-B homolog (Drosophila) NKAIN4 Na+/K+ transporting ATPase interacting 4 NKD1 naked cuticle homolog 1 (Drosophila) NNMT nicotinamide N-methyltransferase NOD1 nucleotide-binding oligomerization domain containing 1 NPR1 natriuretic peptide receptor A/guanylate cyclase A (atrionatriuretic peptide receptor A) NR1D1 nuclear receptor subfamily 1, group D, member 1 NR3C1 nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor) NR4A1 nuclear receptor subfamily 4, group A, member 1 NRGN neurogranin (protein kinase C substrate, RC3) NUCKS1 nuclear casein kinase and cyclin-dependent kinase substrate 1 OAT ornithine aminotransferase (gyrate atrophy) OGDH oxoglutarate (alpha-ketoglutarate) dehydrogenase (lipoamide) OGN osteoglycin OPA3 optic atrophy 3 (autosomal recessive, with chorea and spastic paraplegia) ORAI3 ORAI calcium release-activated calcium modulator 3 OSR1 odd-skipped related 1 (Drosophila) OXCT1 3-oxoacid CoA transferase 1 OXNAD1 oxidoreductase NAD-binding domain containing 1 PARD3B par-3 partitioning defective 3 homolog B (C. elegans) PARP14 poly (ADP-ribose) polymerase family, member 14 PARP4 poly (ADP-ribose) polymerase family, member 4 PARVB parvin, beta PBX1 pre-B-cell leukemia homeobox 1 PCDH15 protocadherin 15 PCDHGB5 protocadherin gamma subfamily B, 5 PCM1 pericentriolar material 1 PDAP1 PDGFA associated protein 1; similar to PDGFA associated protein 1 PDCD6IP programmed cell death 6 interacting protein PDE4DIP hypothetical protein LOC100134230; similar to KIAA0454 protein; similar to phosphodiesterase 4D interacting protein isoform 2; phosphodiesterase 4D interacting protein PDIA3 protein disulfide isomerase family A, member 3 PDIA4 protein disulfide isomerase family A, member 4 PDPN podoplanin PEF1 penta-EF-hand domain containing 1 PELI1 pellino homolog 1 (Drosophila) PER1 period homolog 1 (Drosophila) PF4 platelet factor 4 PFN1 profilin 1 PGCP plasma glutamate carboxypeptidase PGRMC1 progesterone receptor membrane component 1 PHF21A PHD finger protein 21A PHF3 PHD finger protein 3 PHIP pleckstrin homology domain interacting protein PIGT phosphatidylinositol glycan anchor biosynthesis, class T PIK3C2A phosphoinositide-3-kinase, class 2, alpha polypeptide PIM1 pim-1 oncogene PITPNM2 phosphatidylinositol transfer protein, membrane-associated 2 PKHD1L1 polycystic kidney and hepatic disease 1 (autosomal recessive)-like 1 PKNOX1 PBX/knotted 1 homeobox 1 PLA2G4A phospholipase A2, group IVA (cytosolic, calcium-dependent) PLAT plasminogen activator, tissue PLCE1 phospholipase C, epsilon 1 PLK1S1 non-protein coding RNA 153 PLK2 polo-like kinase 2 (Drosophila) PLOD2 procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 PLXDC1 plexin domain containing 1 PLXDC2 plexin domain containing 2 PLXNA4 plexin A4 PMP22 peripheral myelin protein 22 PNRC1 proline-rich nuclear receptor coactivator 1 PODN podocan PPAP2A phosphatidic acid phosphatase type 2A PPBP pro-platelet basic protein (chemokine (C-X-C motif) ligand 7) PPFIBP2 PTPRF interacting protein, binding protein 2 (liprin beta 2) PPIG peptidylprolyl isomerase G (cyclophilin G) PPL periplakin PPP1CB protein phosphatase 1, catalytic subunit, beta isoform; speedy homolog A (Xenopus laevis) PPP1R12A protein phosphatase 1, regulatory (inhibitor) subunit 12A PPP1R15A protein phosphatase 1, regulatory (inhibitor) subunit 15A PPP3CA protein phosphatase 3 (formerly 2B), catalytic subunit, alpha isoform PPPDE1 PPPDE peptidase domain containing 1 PQLC3 PQ loop repeat containing 3 PRELP proline/arginine-rich end leucine-rich repeat protein PRG4 proteoglycan 4 PRKAR2A protein kinase, cAMP-dependent, regulatory, type II, alpha PRPF40A PRP40 pre-mRNA processing factor 40 homolog A (S. cerevisiae) PRR13 proline rich 13 PRSS23 protease, serine, 23 PSD pleckstrin and Sec7 domain containing PSIP1 PC4 and SFRS1 interacting protein 1 PSMB2 proteasome (prosome, macropain) subunit, beta type, 2 PSMD11 proteasome (prosome, macropain) 26S subunit, non-ATPase, 11 PSMD7 proteasome (prosome, macropain) 26S subunit, non-ATPase, 7 PTGES3 prostaglandin E synthase 3 (cytosolic) PTGIS prostaglandin I2 (prostacyclin) synthase PTGS1 prostaglandin-endoperoxide synthase 1 (prostaglandin G/H synthase and cyclooxygenase) PTMA hypothetical LOC728026; prothymosin, alpha; hypothetical gene supported by BC013859; prothymosin, alpha pseudogene 4 (gene sequence 112) PTP4A2 protein tyrosine phosphatase type IVA, member 2 PTPLAD2 protein tyrosine phosphatase-like A domain containing 2 PTPRD protein tyrosine phosphatase, receptor type, D PTPRF protein tyrosine phosphatase, receptor type, F PTRF polymerase 1 and transcript release factor QRICH1 glutamine-rich 1 QSER1 glutamine and serine rich 1 RAB11FIP1 RAB11 family interacting protein 1 (class I) RAB1B RAB1B, member RAS oncogene family RAB5C RAB5C, member RAS oncogene family RAB6B RAB6B, member RAS oncogene family RABGAP1L RAB GTPase activating protein 1-like RALBP1 hypothetical LOC100129773; ralA binding protein 1 RALY RNA binding protein, autoantigenic (hnRNP-associated with lethal yellow homolog (mouse)) RARRES2 retinoic acid receptor responder (tazarotene induced) 2 RB1CC1 RB1-inducible coiled-coil 1 RBBP6 retinoblastoma binding protein 6 RBBP8 retinoblastoma binding protein 8 RBM25 RNA binding motif protein 25 RBM27 RNA binding motif protein 27 RBM3 RNA binding motif (RNP1, RRM) protein 3 RBPMS RNA binding protein with multiple splicing RDX radixin REST RE1-silencing transcription factor RGMA RGM domain family, member A RGS10 regulator of G-protein signaling 10 RHOB ras homolog gene family, member B RHOJ ras homolog gene family, member J RHOU ras homolog gene family, member U RNASE4 ribonuclease, RNase A family, 4 RND3 Rho family GTPase 3 RNF167 ring finger protein 167 RNF20 ring finger protein 20 ROCK1 similar to Rho-associated, coiled-coil containing protein kinase 1; Rho- associated, coiled-coil containing protein kinase 1 ROCK2 Rho-associated, coiled-coil containing protein kinase 2 RPP25 ribonuclease P/MRP 25 kDa subunit RRAS2 related RAS viral (r-ras) oncogene homolog 2; similar to related RAS viral (r-ras) oncogene homolog 2 RSPO1 R-spondin homolog (Xenopus laevis) RTF1 Rtf1, Paf1/RNA polymerase II complex component, homolog (S. cerevisiae) RTN1 reticulon 1 RYK RYK receptor-like tyrosine kinase SARNP SAP domain containing ribonucleoprotein SAT1 spermidine/spermine N1-acetyltransferase 1 SBSN suprabasin SDC4 syndecan 4 SDPR serum deprivation response (phosphatidylserine binding protein) SEC62 SEC62 homolog (S. cerevisiae) SECISBP2 SECIS binding protein 2 SEMA5A sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic domain, (semaphorin) 5A SENP6 SUMO1/sentrin specific peptidase 6 SEP15 15 kDa selenoprotein SEPT9 septin 9 SERINC5 serine incorporator 5 SERPING1 serpin peptidase inhibitor, clade G (C1 inhibitor), member 1 SERPINH1 serpin peptidase inhibitor, clade H (heat shock protein 47), member 1, (collagen binding protein 1) SESN1 sestrin 1 SETD2 SET domain containing 2 SF3B1 splicing factor 3b, subunit 1, 155 kDa SF3B4 splicing factor 3b, subunit 4, 49 kDa SFRS18 splicing factor, arginine/serine-rich 18 SHC1 SHC (Src homology 2 domain containing) transforming protein 1 SHFM1 split hand/foot malformation (ectrodactyly) type 1 SIAE sialic acid acetylesterase SIRT2 sirtuin (silent mating type information regulation 2 homolog) 2 (S. cerevisiae) SLC10A3 solute carrier family 10 (sodium/bile acid cotransporter family), member 3 SLC16A1 solute carrier family 16, member 1 (monocarboxylic acid transporter 1) SLC1A5 solute carrier family 1 (neutral amino acid transporter), member 5 SLC26A3 solute carrier family 26, member 3 SLC27A3 solute carrier family 27 (fatty acid transporter), member 3 SLC38A1 solute carrier family 38, member 1 SLC39A8 solute carrier family 39 (zinc transporter), member 8 SLC43A3 solute carrier family 43, member 3 SLC4A4 solute carrier family 4, sodium bicarbonate cotransporter, member 4 SLC6A4 solute carrier family 6 (neurotransmitter transporter, serotonin), member 4 SLC6A6 solute carrier family 6 (neurotransmitter transporter, taurine), member 6 SLC8A1 solute carrier family 8 (sodium/calcium exchanger), member 1 SLC9A3R1 solute carrier family 9 (sodium/hydrogen exchanger), member 3 regulator 1 SLPI secretory leukocyte peptidase inhibitor SLTM SAFB-like, transcription modulator SLU7 SLU7 splicing factor homolog (S. cerevisiae) SLURP1 secreted LY6/PLAUR domain containing 1 SMAD4 SMAD family member 4 SMARCA2 SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 2 SMARCA5 SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 5 SMC2 structural maintenance of chromosomes 2 SMC3 structural maintenance of chromosomes 3 SMC4 structural maintenance of chromosomes 4 SMC6 structural maintenance of chromosomes 6 SMCHD1 structural maintenance of chromosomes flexible hinge domain containing 1 SMPD3 sphingomyelin phosphodiesterase 3, neutral membrane (neutral sphingomyelinase II) SNRNP70 small nuclear ribonucleoprotein 70 kDa (U1) SNTB2 syntrophin, beta 2 (dystrophin-associated protein A1, 59 kDa, basic component 2) SOAT1 sterol O-acyltransferase 1 SOCS3 suppressor of cytokine signaling 3 SOD3 superoxide dismutase 3, extracellular SORBS1 sorbin and SH3 domain containing 1 SORBS3 sorbin and SH3 domain containing 3 SOX6 SRY (sex determining region Y)-box 6 SP100 SP100 nuclear antigen SPAG9 sperm associated antigen 9 SPARC secreted protein, acidic, cysteine-rich (osteonectin) SPEN spen homolog, transcriptional regulator (Drosophila) SPINT2 serine peptidase inhibitor, Kunitz type, 2 SPOCK2 sparc/osteonectin, cwcv and kazal-like domains proteoglycan (testican) 2 SPON2 spondin 2, extracellular matrix protein SPOP speckle-type POZ protein SRC v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian) SRRM1 serine/arginine repetitive matrix 1 SSH2 slingshot homolog 2 (Drosophila) SSR3 signal sequence receptor, gamma (translocon-associated protein gamma) ST3GAL1 ST3 beta-galactoside alpha-2,3-sialyltransferase 1 STAG1 stromal antigen 1 STAR steroidogenic acute regulatory protein STARD5 StAR-related lipid transfer (START) domain containing 5 STAT3 signal transducer and activator of transcription 3 (acute-phase response factor) STIM1 stromal interaction molecule 1 STK10 serine/threonine kinase 10 STK40 serine/threonine kinase 40 STMN2 stathmin-like 2 STRA6 stimulated by retinoic acid gene 6 homolog (mouse) STRN3 striatin, calmodulin binding protein 3 SULF1 sulfatase 1 SULF2 sulfatase 2 SUPT16H suppressor of Ty 16 homolog (S. cerevisiae); suppressor of Ty 16 homolog (S. cerevisiae) pseudogene SV2A synaptic vesicle glycoprotein 2A SYNE1 spectrin repeat containing, nuclear envelope 1 SYNE2 spectrin repeat containing, nuclear envelope 2 SYT11 synaptotagmin XI SYTL1 synaptotagmin-like 1 TAF3 TAF3 RNA polymerase II, TATA box binding protein (TBP)-associated factor, 140 kDa TAF7 TAF7 RNA polymerase II, TATA box binding protein (TBP)-associated factor, 55 kDa TAPBP TAP binding protein (tapasin) TBC1D15 TBC1 domain family, member 15 TBCEL tubulin folding cofactor E-like TBL1X transducin (beta)-like 1X-linked TBX18 T-box 18 TCEAL8 transcription elongation factor A (SII)-like 8 TCF7L1 transcription factor 7-like 1 (T-cell specific, HMG-box) TFDP2 transcription factor Dp-2 (E2F dimerization partner 2) TGFB1I1 transforming growth factor beta 1 induced transcript 1 TGFB2 transforming growth factor, beta 2 TGFBR2 transforming growth factor, beta receptor II (70/80 kDa) TGM2 transglutaminase 2 (C polypeptide, protein-glutamine-gamma- glutamyltransferase) THBD thrombomodulin THBS1 thrombospondin 1 THOC2 THO complex 2 THRAP3 thyroid hormone receptor associated protein 3 THSD4 thrombospondin, type I, domain containing 4 TIMP2 TIMP metallopeptidase inhibitor 2 TIRAP toll-interleukin 1 receptor (TIR) domain containing adaptor protein TLR2 toll-like receptor 2 TM4SF1 transmembrane 4 L six family member 1 TM4SF5 transmembrane 4 L six family member 5 TMCC3 transmembrane and coiled-coil domain family 3 TMCO1 transmembrane and coiled-coil domains 1 TMCO7 transmembrane and coiled-coil domains 7 TMED2 transmembrane emp24 domain trafficking protein 2 TMEM119 transmembrane protein 119 TMEM140 transmembrane protein 140 TMEM151A transmembrane protein 151A TMEM221 transmembrane protein 221 TMEM50A transmembrane protein 50A TMEM98 similar to transmembrane protein 98; transmembrane protein 98 TMOD3 tropomodulin 3 (ubiquitous) TMPO thymopoietin TMSB4X thymosin-like 2 (pseudogene); thymosin-like 1 (pseudogene); thymosin beta 4, X-linked TNXB tenascin XB; tenascin XA pseudogene TOB2 transducer of ERBB2, 2 TOPORS topoisomerase 1 binding, arginine/serine-rich TPM3 tropomyosin 3 TPPP3 tubulin polymerization-promoting protein family member 3 TPT1 similar to tumor protein, translationally-controlled 1; tumor protein, translationally-controlled 1 TRAFD1 TRAF-type zinc finger domain containing 1 TRIB1 tribbles homolog 1 (Drosophila) TRIM8 tripartite motif-containing 8 TRPM7 transient receptor potential cation channel, subfamily M, member 7 TSC22D3 TSC22 domain family, member 3; GRAM domain containing 4 TSHZ1 teashirt zinc finger homeobox 1 TSIX XIST antisense RNA (non-protein coding) TSPAN31 tetraspanin 31 TSPAN5 tetraspanin 5 TTC28 chromosome 6 open reading frame 35; hCG1820764; tetratricopeptide repeat domain 28 TTC38 tetratricopeptide repeat domain 38 TUBA1A tubulin, alpha la TUBB2A tubulin, beta 2A TWSG1 twisted gastrulation homolog 1 (Drosophila) TXNDC5 thioredoxin domain containing 5 (endoplasmic reticulum); muted homolog (mouse) TXNRD1 thioredoxin reductase 1; hypothetical LOC100130902 UAP1 UDP-N-acteylglucosamine pyrophosphorylase 1 UBA7 ubiquitin-like modifier activating enzyme 7 UBE2D1 ubiquitin-conjugating enzyme E2D 1 (UBC4/5 homolog, yeast) UBE2L6 ubiquitin-conjugating enzyme E2L 6 UBE2N ubiquitin-conjugating enzyme E2N (UBC13 homolog, yeast) UBE2V1 ubiquitin-conjugating enzyme E2 variant 1; ubiquitin-conjugating enzyme E2 variant 1 pseudogene 2; transmembrane protein 189; TMEM189-UBE2V1 readthrough transcript UBQLN2 ubiquilin 2 UBXN2A UBX domain protein 2A UBXN4 UBX domain protein 4 UGDH UDP-glucose dehydrogenase UPK1B uroplakin 1B UPK3B uroplakin 3B USP16 ubiquitin specific peptidase 16 USP2 ubiquitin specific peptidase 2 USP25 ubiquitin specific peptidase 25 USP54 ubiquitin specific peptidase 54 USP8 ubiquitin specific peptidase 8 UTP20 similar to Down-regulated in metastasis protein (Key-1A6 protein) (Novel nucleolar protein 73) (NNP73); UTP20, small subunit (SSU) processome component, homolog (yeast) VAT1 vesicle amine transport protein 1 homolog (T. californica) VIM vimentin VPS13A vacuolar protein sorting 13 homolog A (S. cerevisiae) VWA5A von Willebrand factor A domain containing 5A WAC WW domain containing adaptor with coiled-coil WASF2 WAS protein family, member 2 WDR26 WD repeat domain 26 WDR92 WD repeat domain 92 WFDC1 WAP four-disulfide core domain 1 WLS G protein-coupled receptor 177 WNT4 wingless-type MMTV integration site family, member 4 WRNIP1 Werner helicase interacting protein 1 WT1 Wilms tumor 1 WWC2 WW and C2 domain containing 2 XDH xanthine dehydrogenase XIST X (inactive)-specific transcript (non-protein coding) YIPF5 Yip1 domain family, member 5 YWHAZ tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide ZBTB16 zinc finger and BTB domain containing 16 ZBTB20 zinc finger and BTB domain containing 20 ZBTB4 zinc finger and BTB domain containing 4 ZBTB7C zinc finger and BTB domain containing 7C ZC3H13 zinc finger CCCH-type containing 13 ZC3H18 zinc finger CCCH-type containing 18 ZCCHC11 zinc finger, CCHC domain containing 11 ZCCHC3 zinc finger, CCHC domain containing 3 ZFAND6 zinc finger, AN1-type domain 6 ZFHX4 zinc finger homeobox 4 ZFP36 zinc finger protein 36, C3H type, homolog (mouse) ZMAT1 zinc finger, matrin type 1 ZRSR1 zinc finger (CCCH type), RNA-binding motif and serine/arginine rich 1 ZZEF1 zinc finger, ZZ-type with EF-hand domain 1

The gene names listed in Table 7 and Table 8 are common names. NCBI Gene ID numbers for each of the genes listed in Table 7 or Table 8 can be obtained by searching the “Gene” Database of the NCBI (available on the World Wide Web at http://www.ncbi.nlm.nih.gov/) using the common name as the query and selecting the first returned Homo sapiens (for the genes in Table 8) or Mus musculus gene (for the genes in Table 7). Other genes may be obtained using the UCSC genome browser (available on the World Wide Web at http://genome.ucsc.edu) using the Gene Sorter function. Human homologs of mouse genes can be readily identified, e.g. the identified homologs in the NCBI database, or by querying databases such as BLAST. In certain embodiments, the marker gene(s) are selected from the genes listed in Table 7, Table 8, or Table 14.

In a CTC, the marker genes listed in Table 7, Table 8, or Table 14 can be upregulated, e.g. for marker genes listed in Table 7, Table 8, or Table 14, if the measured marker gene expression in a cell or sample is higher as compared to a reference level of that marker gene's expression, then the cell is identified as a CTC and/or the sample is identified as comprising CTCs. Preferably, once looks at a statistically significant change. However, even if a few genes in a group do not differ from normal, a sample can be identified as comprising CTCs if the overall change of the group shows a significant change, preferably a statistically significant change. All possible combinations of 2 or more of the indicated markers are contemplated herein.

The level of a gene expression product of a marker gene in Table 7, Table 8, or Table 14 which is higher than a reference level of that marker gene by at least about 10% than the reference amount, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 80%, at least about 100%, at least about 200%, at least about 300%, at least about 500% or at least about 1000% or more, is indicative of the presence of a CTC.

In some embodiments, the reference can be a level of expression of the marker gene product in a cell or population of cells which are not CTCs, e.g. the average level in non-circulating tumor cells and/or circulating cells which are not cancer cells. In some embodiments, the reference can also be a level of expression of the marker gene product in a control sample, a pooled sample of control individuals or a numeric value or range of values based on the same.

In some embodiments, the methods and assays described herein include (a) transforming the gene expression product into a detectable gene target; (b) measuring the amount of the detectable gene target; and (c) comparing the amount of the detectable gene target to an amount of a reference, wherein if the amount of the detectable gene target is statistically significantly different than the amount of the reference level, the presence and/or level of CTCs is determined. In some embodiments, if the amount of the detectable gene target is not statistically significantly different than the amount of the reference level, the sample is identified as not comprising CTCs.

As used herein, the term “transforming” or “transformation” refers to changing an object or a substance, e.g., biological sample, nucleic acid or protein, into another substance. The transformation can be physical, biological or chemical. Exemplary physical transformation includes, but not limited to, pre-treatment of a biological sample, e.g., from whole blood to blood serum by differential centrifugation. A biological/chemical transformation can involve at least one enzyme and/or a chemical reagent in a reaction. For example, a DNA sample can be digested into fragments by one or more restriction enzyme, or an exogenous molecule can be attached to a fragmented DNA sample with a ligase. In some embodiments, a DNA sample can undergo enzymatic replication, e.g., by polymerase chain reaction (PCR).

Methods to measure gene expression products associated with the marker genes described herein are well known to a skilled artisan. Such methods to measure gene expression products, e.g., protein level, include ELISA (enzyme linked immunosorbent assay), western blot, FACS, radioimmunological assay; (RIA); sandwich assay; fluorescent in situ hybridization (FISH); immunohistological staining; immunoelectrophoresis; immunoprecipitation, and immunofluorescence using detection reagents such as an antibody or protein binding agents. Alternatively, a peptide can be detected in a subject by introducing into a subject a labeled anti-peptide antibody and other types of detection agent. For example, the antibody can be labeled with a radioactive marker whose presence and location in the subject is detected by standard imaging techniques.

For example, antibodies for the polypeptide expression products of the marker genes described herein are commercially available and can be used for the purposes of the invention to measure protein expression levels, e.g. anti-IGFBP5 (Cat. No. 4255; Abcam; Cambridge, Mass.). Alternatively, since the amino acid sequences for the marker genes described herein are known and publically available at NCBI website, one of skill in the art can raise their own antibodies against these proteins of interest for the purpose of the invention. The amino acid sequences of the marker genes described herein have been assigned NCBI accession numbers for different species such as human, mouse and rat.

In some embodiments, immunohistochemistry (“IHC”) and immunocytochemistry (“ICC”) techniques can be used. IHC is the application of immunochemistry to tissue sections, whereas ICC is the application of immunochemistry to cells or tissue imprints after they have undergone specific cytological preparations such as, for example, liquid-based preparations. Immunochemistry is a family of techniques based on the use of an antibody, wherein the antibodies are used to specifically target molecules inside or on the surface of cells. The antibody typically contains a marker that will undergo a biochemical reaction, and thereby experience a change color, upon encountering the targeted molecules. In some instances, signal amplification can be integrated into the particular protocol, wherein a secondary antibody, that includes the marker stain or marker signal, follows the application of a primary specific antibody.

In some embodiments, the assay can be a Western blot analysis. Alternatively, proteins can be separated by two-dimensional gel electrophoresis systems. Two-dimensional gel electrophoresis is well known in the art and typically involves iso-electric focusing along a first dimension followed by SDS-PAGE electrophoresis along a second dimension. These methods also require a considerable amount of cellular material. The analysis of 2D SDS-PAGE gels can be performed by determining the intensity of protein spots on the gel, or can be performed using immune detection. In other embodiments, protein samples are analyzed by mass spectroscopy.

Immunological tests can be used with the methods and assays described herein and include, for example, competitive and non-competitive assay systems using techniques such as Western blots, radioimmunoassay (RIA), ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays, immunoprecipitation assays, immunodiffusion assays, agglutination assays, e.g. latex agglutination, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, e.g. FIA (fluorescence-linked immunoassay), chemiluminescence immunoassays (CLIA), electrochemiluminescence immunoassay (ECLIA, counting immunoassay (CIA), lateral flow tests or immunoassay (LFIA), magnetic immunoassay (MIA), and protein A immunoassays. Methods for performing such assays are known in the art, provided an appropriate antibody reagent is available. In some embodiment, the immunoassay can be a quantitative or a semi-quantitative immunoassay.

An immunoassay is a biochemical test that measures the concentration of a substance in a biological sample, typically a fluid sample such as serum, using the interaction of an antibody or antibodies to its antigen. The assay takes advantage of the highly specific binding of an antibody with its antigen. For the methods and assays described herein, specific binding of the target polypeptides with respective proteins or protein fragments, or an isolated peptide, or a fusion protein described herein occurs in the immunoassay to form a target protein/peptide complex. The complex is then detected by a variety of methods known in the art. An immunoassay also often involves the use of a detection antibody.

Enzyme-linked immunosorbent assay, also called ELISA, enzyme immunoassay or EIA, is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality control check in various industries.

In one embodiment, an ELISA involving at least one antibody with specificity for the particular desired antigen (i.e. a marker gene polypeptide as described herein) can also be performed. A known amount of sample and/or antigen is immobilized on a solid support (usually a polystyrene micro titer plate). Immobilization can be either non-specific (e.g., by adsorption to the surface) or specific (e.g. where another antibody immobilized on the surface is used to capture antigen or a primary antibody). After the antigen is immobilized, the detection antibody is added, forming a complex with the antigen. The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody which is linked to an enzyme through bio-conjugation. Between each step the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. After the final wash step the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample. Older ELISAs utilize chromogenic substrates, though newer assays employ fluorogenic substrates with much higher sensitivity.

In another embodiment, a competitive ELISA is used. Purified antibodies that are directed against a target polypeptide or fragment thereof are coated on the solid phase of multi-well plate, i.e., conjugated to a solid surface. A second batch of purified antibodies that are not conjugated on any solid support is also needed. These non-conjugated purified antibodies are labeled for detection purposes, for example, labeled with horseradish peroxidase to produce a detectable signal. A sample (e.g., tumor, blood, serum or urine) from a subject is mixed with a known amount of desired antigen (e.g., a known volume or concentration of a sample comprising a target polypeptide) together with the horseradish peroxidase labeled antibodies and the mixture is then are added to coated wells to form competitive combination. After incubation, if the polypeptide level is high in the sample, a complex of labeled antibody reagent-antigen will form. This complex is free in solution and can be washed away. Washing the wells will remove the complex. Then the wells are incubated with TMB (3, 3′, 5, 5′-tetramethylbenzidene) color development substrate for localization of horseradish peroxidase-conjugated antibodies in the wells. There will be no color change or little color change if the target polypeptide level is high in the sample. If there is little or no target polypeptide present in the sample, a different complex in formed, the complex of solid support bound antibody reagents-target polypeptide. This complex is immobilized on the plate and is not washed away in the wash step. Subsequent incubation with TMB will produce much color change. Such a competitive ELSA test is specific, sensitive, reproducible and easy to operate.

There are other different forms of ELISA, which are well known to those skilled in the art. The standard techniques known in the art for ELISA are described in “Methods in Immunodiagnosis”, 2nd Edition, Rose and Bigazzi, eds. John Wiley & Sons, 1980; and Oellerich, M. 1984, J. Clin. Chem. Clin. Biochem. 22:895-904. These references are hereby incorporated by reference in their entirety.

In one embodiment, the levels of a polypeptide in a sample can be detected by a lateral flow immunoassay test (LFIA), also known as the immunochromatographic assay, or strip test. LFIAs are a simple device intended to detect the presence (or absence) of antigen, e.g. a polypeptide, in a fluid sample. There are currently many LFIA tests are used for medical diagnostics either for home testing, point of care testing, or laboratory use. LFIA tests are a form of immunoassay in which the test sample flows along a solid substrate via capillary action. After the sample is applied to the test strip it encounters a colored reagent (generally comprising antibody specific for the test target antigen) bound to microparticles which mixes with the sample and transits the substrate encountering lines or zones which have been pretreated with another antibody or antigen. Depending upon the level of target polypeptides present in the sample the colored reagent can be captured and become bound at the test line or zone. LFIAs are essentially immunoassays adapted to operate along a single axis to suit the test strip format or a dipstick format. Strip tests are extremely versatile and can be easily modified by one skilled in the art for detecting an enormous range of antigens from fluid samples such as urine, blood, water, and/or homogenized tumor samples etc. Strip tests are also known as dip stick test, the name bearing from the literal action of “dipping” the test strip into a fluid sample to be tested. LFIA strip tests are easy to use, require minimum training and can easily be included as components of point-of-care test (POCT) diagnostics to be use on site in the field. LFIA tests can be operated as either competitive or sandwich assays. Sandwich LFIAs are similar to sandwich ELISA. The sample first encounters colored particles which are labeled with antibodies raised to the target antigen. The test line will also contain antibodies to the same target, although it may bind to a different epitope on the antigen. The test line will show as a colored band in positive samples. In some embodiments, the lateral flow immunoassay can be a double antibody sandwich assay, a competitive assay, a quantitative assay or variations thereof. Competitive LFIAs are similar to competitive ELISA. The sample first encounters colored particles which are labeled with the target antigen or an analogue. The test line contains antibodies to the target/its analogue. Unlabelled antigen in the sample will block the binding sites on the antibodies preventing uptake of the colored particles. The test line will show as a colored band in negative samples. There are a number of variations on lateral flow technology. It is also possible to apply multiple capture zones to create a multiplex test.

The use of “dip sticks” or LFIA test strips and other solid supports have been described in the art in the context of an immunoassay for a number of antigen biomarkers. U.S. Pat. Nos. 4,943,522; 6,485,982; 6,187,598; 5,770,460; 5,622,871; 6,565,808, U.S. patent application Ser. No. 10/278,676; U.S. Ser. No. 09/579,673 and U.S. Ser. No. 10/717,082, which are incorporated herein by reference in their entirety, are non-limiting examples of such lateral flow test devices. Examples of patents that describe the use of “dip stick” technology to detect soluble antigens via immunochemical assays include, but are not limited to U.S. Pat. Nos. 4,444,880; 4,305,924; and 4,135,884; which are incorporated by reference herein in their entireties. The apparatuses and methods of these three patents broadly describe a first component fixed to a solid surface on a “dip stick” which is exposed to a solution containing a soluble antigen that binds to the component fixed upon the “dip stick,” prior to detection of the component-antigen complex upon the stick. It is within the skill of one in the art to modify the teachings of this “dip stick” technology for the detection of polypeptides using antibody reagents as described herein.

Other techniques can be used to detect the level of a polypeptide in a sample. One such technique is the dot blot, an adaptation of Western blotting (Towbin et al., Proc. Nat. Acad. Sci. 76:4350 (1979)). In a Western blot, the polypeptide or fragment thereof can be dissociated with detergents and heat, and separated on an SDS-PAGE gel before being transferred to a solid support, such as a nitrocellulose or PVDF membrane. The membrane is incubated with an antibody reagent specific for the target polypeptide or a fragment thereof. The membrane is then washed to remove unbound proteins and proteins with non-specific binding. Detectably labeled enzyme-linked secondary or detection antibodies can then be used to detect and assess the amount of polypeptide in the sample tested. The intensity of the signal from the detectable label corresponds to the amount of enzyme present, and therefore the amount of polypeptide. Levels can be quantified, for example by densitometry.

Flow cytometry is a well-known technique for analyzing and sorting cells (or other small particles) suspended in a fluid stream. This technique allows simultaneous analysis of the physical and/or chemical characteristics of single cells flowing through an optical, electronic, or magnetic detection apparatus. As applied to FACS, the flow cytometer consists of a flow cell which carries the cells in a fluid stream in single file through a light source with excites the fluorescently labeled detection marker(s) (for example, antibody reagents) and measures the fluorescent character of the cell. The fluid stream is then ejected through a nozzle and a charging ring, under pressure, which breaks the fluid into droplets. The flow cell device and fluid stream is calibrated such that there is a relatively large distance between individual cells or bound groups of cells, resulting in a low probability that any droplet contains more than a single cell or bound group of cells. The charging ring charges the droplets based on the fluorescence characteristic of the cell which is contained therein. The charged droplets are then deflected by an electrostatically-charged deflection system which diverts the droplets into various containers based upon their charge (related to the fluorescence intensity of the cell). A FACS system (e.g. the FACSARIA™ flow cytometer (BD Biosciences) and FLOWJO™ Version 7.6.4 (TreeStar)) can detect and record the number of total cells as well as the number of cells which display one or more fluorescent characteristics, e.g. the total number of cells bound by one or more antibody reagents specific for a CTC marker gene.

In certain embodiments, the gene expression products as described herein can be instead determined by determining the level of messenger RNA (mRNA) expression of genes associated with the marker genes described herein. Such molecules can be isolated, derived, or amplified from a biological sample, such as a tumor biopsy. Detection of mRNA expression is known by persons skilled in the art, and comprise, for example but not limited to, PCR procedures, RT-PCR, quantitative PCR or RT-PCR, Northern blot analysis, differential gene expression, RNA protection assay, microarray analysis, hybridization methods, next-generation sequencing etc. Non-limiting examples of next-generation sequencing technologies can include Ion Torrent, Illumina, SOLiD, 454; Massively Parallel Signature Sequencing solid-phase, reversible dye-terminator sequencing; and DNA nanoball sequencing.

In general, the PCR procedure describes a method of gene amplification which is comprised of (i) sequence-specific hybridization of primers to specific genes or sequences within a nucleic acid sample or library, (ii) subsequent amplification involving multiple rounds of annealing, elongation, and denaturation using a thermostable DNA polymerase, and (iii) screening the PCR products for a band of the correct size. The primers used are oligonucleotides of sufficient length and appropriate sequence to provide initiation of polymerization, i.e. each primer is specifically designed to be complementary to a strand of the genomic locus to be amplified. In an alternative embodiment, mRNA level of gene expression products described herein can be determined by reverse-transcription (RT) PCR and by quantitative RT-PCR (QRT-PCR) or real-time PCR methods. Methods of RT-PCR and QRT-PCR are well known in the art. The nucleic acid sequences of the marker genes described herein have been assigned NCBI accession numbers for different species such as human, mouse and rat. Accordingly, a skilled artisan can design an appropriate primer based on the known sequence for determining the mRNA level of the respective gene.

Nucleic acid and ribonucleic acid (RNA) molecules can be isolated from a particular biological sample using any of a number of procedures, which are well-known in the art, the particular isolation procedure chosen being appropriate for the particular biological sample. For example, freeze-thaw and alkaline lysis procedures can be useful for obtaining nucleic acid molecules from solid materials; heat and alkaline lysis procedures can be useful for obtaining nucleic acid molecules from urine; and proteinase K extraction can be used to obtain nucleic acid from blood (Roiff, A et al. PCR: Clinical Diagnostics and Research, Springer (1994)).

In general, the PCR procedure describes a method of gene amplification which is comprised of (i) sequence-specific hybridization of primers to specific genes within a nucleic acid sample or library, (ii) subsequent amplification involving multiple rounds of annealing, elongation, and denaturation using a DNA polymerase, and (iii) screening the PCR products for a band of the correct size. The primers used are oligonucleotides of sufficient length and appropriate sequence to provide initiation of polymerization, i.e. each primer is specifically designed to be complementary to each strand of the nucleic acid molecule to be amplified.

In an alternative embodiment, mRNA level of gene expression products described herein can be determined by reverse-transcription (RT) PCR and by quantitative RT-PCR (QRT-PCR) or real-time PCR methods. Methods of RT-PCR and QRT-PCR are well known in the art.

In some embodiments, one or more of the reagents (e.g. an antibody reagent and/or nucleic acid probe) described herein can comprise a detectable label and/or comprise the ability to generate a detectable signal (e.g. by catalyzing reaction converting a compound to a detectable product). Detectable labels can comprise, for example, a light-absorbing dye, a fluorescent dye, or a radioactive label. Detectable labels, methods of detecting them, and methods of incorporating them into reagents (e.g. antibodies and nucleic acid probes) are well known in the art.

In some embodiments, detectable labels can include labels that can be detected by spectroscopic, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, or chemical means, such as fluorescence, chemifluoresence, or chemiluminescence, or any other appropriate means. The detectable labels used in the methods described herein can be primary labels (where the label comprises a moiety that is directly detectable or that produces a directly detectable moiety) or secondary labels (where the detectable label binds to another moiety to produce a detectable signal, e.g., as is common in immunological labeling using secondary and tertiary antibodies). The detectable label can be linked by covalent or non-covalent means to the reagent. Alternatively, a detectable label can be linked such as by directly labeling a molecule that achieves binding to the reagent via a ligand-receptor binding pair arrangement or other such specific recognition molecules. Detectable labels can include, but are not limited to radioisotopes, bioluminescent compounds, chromophores, antibodies, chemiluminescent compounds, fluorescent compounds, metal chelates, and enzymes.

In other embodiments, the detection reagent is label with a fluorescent compound. When the fluorescently labeled antibody is exposed to light of the proper wavelength, its presence can then be detected due to fluorescence. In some embodiments, a detectable label can be a fluorescent dye molecule, or fluorophore including, but not limited to fluorescein, phycoerythrin, phycocyanin, o-phthaldehyde, fluorescamine, Cy3™, Cy5™, allophycocyanine, Texas Red, peridenin chlorophyll, cyanine, tandem conjugates such as phycoerythrin-Cy5™, green fluorescent protein, rhodamine, fluorescein isothiocyanate (FITC) and Oregon Green™, rhodamine and derivatives (e.g., Texas red and tetrarhodimine isothiocynate (TRITC)), biotin, phycoerythrin, AMCA, CyDyes™, 6-carboxyfhiorescein (commonly known by the abbreviations FAM and F), 6-carboxy-2′,4′,7′,4,7-hexachlorofiuorescein (HEX), 6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfiuorescein (JOE or J), N,N,N′,N′-tetramethyl-6carboxyrhodamine (TAMRA or T), 6-carboxy-X-rhodamine (ROX or R), 5-carboxyrhodamine-6G (R6G5 or G5), 6-carboxyrhodamine-6G (R6G6 or G6), and rhodamine 110; cyanine dyes, e.g. Cy3, Cy5 and Cy7 dyes; coumarins, e.g umbelliferone; benzimide dyes, e.g. Hoechst 33258; phenanthridine dyes, e.g. Texas Red; ethidium dyes; acridine dyes; carbazole dyes; phenoxazine dyes; porphyrin dyes; polymethine dyes, e.g. cyanine dyes such as Cy3, Cy5, etc; BODIPY dyes and quinoline dyes. In some embodiments, a detectable label can be a radiolabel including, but not limited to ³H, ¹²⁵I, ³⁵S, ¹⁴C, ³²P, and ³³P. In some embodiments, a detectable label can be an enzyme including, but not limited to horseradish peroxidase and alkaline phosphatase. An enzymatic label can produce, for example, a chemiluminescent signal, a color signal, or a fluorescent signal. Enzymes contemplated for use to detectably label an antibody reagent include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. In some embodiments, a detectable label is a chemiluminescent label, including, but not limited to lucigenin, luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester. In some embodiments, a detectable label can be a spectral colorimetric label including, but not limited to colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, and latex) beads.

In some embodiments, detection reagents can also be labeled with a detectable tag, such as c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS, or biotin. Other detection systems can also be used, for example, a biotin-streptavidin system. In this system, the antibodies immunoreactive (i. e. specific for) with the biomarker of interest is biotinylated. Quantity of biotinylated antibody bound to the biomarker is determined using a streptavidin-peroxidase conjugate and a chromagenic substrate. Such streptavidin peroxidase detection kits are commercially available, e. g. from DAKO; Carpinteria, Calif. A reagent can also be detectably labeled using fluorescence emitting metals such as ¹⁵²Eu, or others of the lanthanide series. These metals can be attached to the reagent using such metal chelating groups as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

In some embodiments of any of the aspects described herein, the level of expression products of more than one gene can be determined simultaneously (e.g. a multiplex assay) or in parallel. In some embodiments, the level of expression products of no more than 200 other genes is determined. In some embodiments, the level of expression products of no more than 100 other genes is determined. In some embodiments, the level of expression products of no more than 20 other genes is determined. In some embodiments, the level of expression products of no more than 10 other genes is determined.

The term “sample” or “test sample” as used herein denotes a sample taken or isolated from a biological organism, e.g., a tumor sample from a subject. Exemplary biological samples include, but are not limited to, a biofluid sample; serum; plasma; urine; saliva; a tumor sample; a tumor biopsy and/or tissue sample etc. The term also includes a mixture of the above-mentioned samples. The term “test sample” also includes untreated or pretreated (or pre-processed) biological samples. In some embodiments, a test sample can comprise cells from subject. In some embodiments, a test sample can be a tumor cell test sample, e.g. the sample can comprise cancerous cells, cells from a tumor, and/or a tumor biopsy. In some embodiments, the test sample can be a blood sample.

The test sample can be obtained by removing a sample of cells from a subject, but can also be accomplished by using previously isolated cells (e.g. isolated at a prior timepoint and isolated by the same or another person). In addition, the test sample can be freshly collected or a previously collected sample.

In some embodiments, the test sample can be an untreated test sample. As used herein, the phrase “untreated test sample” refers to a test sample that has not had any prior sample pre-treatment except for dilution and/or suspension in a solution. Exemplary methods for treating a test sample include, but are not limited to, centrifugation, filtration, sonication, homogenization, heating, freezing and thawing, and combinations thereof. In some embodiments, the test sample can be a frozen test sample, e.g., a frozen tissue. The frozen sample can be thawed before employing methods, assays and systems described herein. After thawing, a frozen sample can be centrifuged before being subjected to methods, assays and systems described herein. In some embodiments, the test sample is a clarified test sample, for example, by centrifugation and collection of a supernatant comprising the clarified test sample. In some embodiments, a test sample can be a pre-processed test sample, for example, supernatant or filtrate resulting from a treatment selected from the group consisting of centrifugation, filtration, thawing, purification, and any combinations thereof. In some embodiments, the test sample can be treated with a chemical and/or biological reagent. Chemical and/or biological reagents can be employed to protect and/or maintain the stability of the sample, including biomolecules (e.g., nucleic acid and protein) therein, during processing. One exemplary reagent is a protease inhibitor, which is generally used to protect or maintain the stability of protein during processing. The skilled artisan is well aware of methods and processes appropriate for pre-processing of biological samples required for determination of the level of an expression product as described herein.

In some embodiments, the methods, assays, and systems described herein can further comprise a step of obtaining a test sample from a subject. In some embodiments, the subject can be a human subject.

In some embodiments, the methods and assays described herein can further comprise a step of isolating CTCs or potential CTCs from a sample prior to measuring the level the expression product of one or more of the marker genes described herein. By way of non-limitng example, CTCs can be isolated from, e.g. a blood sample by hydrodynamic size-based separation and/or immunodepletetion of other cell types present in blood samples. The CTC-iChip, described in the Examples herein combines these two approaches to isolate CTCs.

Subjects with high, or at least detectable, levels of CTCs are most likely to benefit from treatment with therapies that specifically target CTCs. Accordingly, provided herein is a method of determining if a subject is likely to respond to treatment with a CTC marker gene-targeted therapy, the method comprising: measuring the level of a CTC marker gene expression product present in the blood and/or stroma of a cancer; and determining that the subject is likely to respond to the treatment if the level of the expression product is increased relative to a reference level. CTC marker gene-targeted therapies are discussed below herein.

Decreased levels of CTCs after administration of a therapy can be indicative of an improvement in the condition of the subject, e.g. the cancer is reduced in size, growth, and/or metastatic potential. Accordingly, provided herein is a method of monitoring the treatment of a subject, the method comprising administering a cancer therapy to a subject in need thereof; measuring the level of a CTC marker gene expression product present in the blood and/or stroma of a cancer; and determining that the subject is responding if the level of the CTC marker gene expression product is decreased relative to the reference level and determining that the subject is not responding to the treatment if the CTC marker gene expression product is not decreased relative to the reference level. In some embodiments the therapy is a chemotherapy, surgical therapy, and/or radiation therapy. In some embodiments, the therapy is a CTC marker gene-targeted therapy. In some embodiments, the reference level is the level of the gene expression product in the patient prior to the administering step.

The CTC marker genes described herein can be targeted directly and/or used to physically target a chemotherapeutic agent to reduce the levels and/or pathogenic activity of CTCs (e.g. metastatic activity). Accordingly, described herein is a method of treating cancer in a subject, the method comprising administering a therapeutically effective amount of a CTC marker gene-targeted therapy to the subject. In some embodiments, the subject is a subject determined to have an elevated level of CTCs and/or an elevated level of a CTC marker gene present in the blood and/or stroma of the cancer.

In some embodiments, the CTC marker gene-targeted therapy can comprise an inhibitor of a CTC marker gene, e.g. the CTC marker gene-targeted therapy can inhibit the level and/or activity of a CTC marker gene. As used herein, the term “inhibitor” refers to an agent which can decrease the expression and/or activity of the targeted expression product (e.g. mRNA encoding the target or a target polypeptide), e.g. by at least 10% or more, e.g. by 10% or more, 50% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 98% or more. The efficacy of an inhibitor of a CTC marker gene, e.g. its ability to decrease the level and/or activity of the CTC marker gene can be determined, e.g. by measuring the level of an expression product and/or the activity of the CTC marker gene. Methods for measuring the level of a given mRNA and/or polypeptide are known to one of skill in the art, e.g. RTPCR with primers can be used to determine the level of RNA and Western blotting with an antibody can be used to determine the level of a polypeptide. The activity of, e.g. a CTC marker gene can be determined, e.g. by measuring the levels and/or survival of CTCs using methods known in the art and described elsewhere herein. In some embodiments, the inhibitor of a CTC marker gene can be an inhibitory nucleic acid; an aptamer; an antibody reagent; an antibody; or a small molecule.

In some embodiments, the inhibitor of a CTC marker gene can be an antibody reagent. As used herein an “antibody” refers to IgG, IgM, IgA, IgD or IgE molecules or antigen-specific antibody fragments thereof (including, but not limited to, a Fab, F(ab′)₂, Fv, disulphide linked Fv, scFv, single domain antibody, closed conformation multispecific antibody, disulphide-linked scfv, diabody), whether derived from any species that naturally produces an antibody, or created by recombinant DNA technology; whether isolated from serum, B-cells, hybridomas, transfectomas, yeast or bacteria.

As described herein, an “antigen” is a molecule that is bound by a binding site on an antibody agent. Typically, antigens are bound by antibody ligands and are capable of raising an antibody response in vivo. An antigen can be a polypeptide, protein, nucleic acid or other molecule or portion thereof. The term “antigenic determinant” refers to an epitope on the antigen recognized by an antigen-binding molecule, and more particularly, by the antigen-binding site of said molecule.

As used herein, the term “antibody reagent” refers to a polypeptide that includes at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and which specifically binds a given antigen. An antibody reagent can comprise an antibody or a polypeptide comprising an antigen-binding domain of an antibody. In some embodiments, an antibody reagent can comprise a monoclonal antibody or a polypeptide comprising an antigen-binding domain of a monoclonal antibody. For example, an antibody can include a heavy (H) chain variable region (abbreviated herein as VH), and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term “antibody reagent” encompasses antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab and sFab fragments, F(ab′)2, Fd fragments, Fv fragments, scFv, and domain antibodies (dAb) fragments (see, e.g. de Wildt et al., Eur J. Immunol. 1996; 26(3):629-39; which is incorporated by reference herein in its entirety)) as well as complete antibodies. An antibody can have the structural features of IgA, IgG, IgE, IgD, IgM (as well as subtypes and combinations thereof). Antibodies can be from any source, including mouse, rabbit, pig, rat, and primate (human and non-human primate) and primatized antibodies. Antibodies also include midibodies, humanized antibodies, chimeric antibodies, and the like.

The VH and VL regions can be further subdivided into regions of hypervariability, termed “complementarity determining regions” (“CDR”), interspersed with regions that are more conserved, termed “framework regions” (“FR”). The extent of the framework region and CDRs has been precisely defined (see, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; which are incorporated by reference herein in their entireties). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

The terms “antigen-binding fragment” or “antigen-binding domain”, which are used interchangeably herein are used to refer to one or more fragments of a full length antibody that retain the ability to specifically bind to a target of interest. Examples of binding fragments encompassed within the term “antigen-binding fragment” of a full length antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546; which is incorporated by reference herein in its entirety), which consists of a VH or VL domain; and (vi) an isolated complementarity determining region (CDR) that retains specific antigen-binding functionality. As used herein, the term “specific binding” refers to a chemical interaction between two molecules, compounds, cells and/or particles wherein the first entity binds to the second, target entity with greater specificity and affinity than it binds to a third entity which is a non-target. In some embodiments, specific binding can refer to an affinity of the first entity for the second target entity which is at least 10 times, at least 50 times, at least 100 times, at least 500 times, at least 1000 times or greater than the affinity for the third nontarget entity.

Additionally, and as described herein, a recombinant humanized antibody can be further optimized to decrease potential immunogenicity, while maintaining functional activity, for therapy in humans. In this regard, functional activity means a polypeptide capable of displaying one or more known functional activities associated with a recombinant antibody or antibody reagent thereof as described herein. Such functional activities include, e.g. the ability to bind to a given CTC marker gene.

In some embodiments, the inhibitor of a CTC marker gene can be an inhibitory nucleic acid reagent. In some embodiments, the inhibitory nucleic acid is an inhibitory RNA (iRNA). Double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). The inhibitory nucleic acids described herein can include an RNA strand (the antisense strand) having a region which is 30 nucleotides or less in length, i.e., 15-30 nucleotides in length, generally 19-24 nucleotides in length, which region is substantially complementary to at least part the targeted mRNA transcript. The use of these iRNAs enables the targeted degradation of mRNA transcripts, resulting in decreased expression and/or activity of the target.

As used herein, the term “iRNA” refers to an agent that contains RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. In one embodiment, an iRNA as described herein effects inhibition of the expression and/or activity of the target mRNA. In certain embodiments, contacting a cell with the inhibitor (e.g. an iRNA) results in a decrease in the target mRNA level in a cell by at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, up to and including 100% of the target mRNA level found in the cell without the presence of the iRNA.

In some embodiments, the iRNA can be a dsRNA. A dsRNA includes two RNA strands that are sufficiently complementary to hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence. The target sequence can be derived from the sequence of an mRNA formed during the expression of the target. The other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. Generally, the duplex structure is between 15 and 30 inclusive, more generally between 18 and 25 inclusive, yet more generally between 19 and 24 inclusive, and most generally between 19 and 21 base pairs in length, inclusive. Similarly, the region of complementarity to the target sequence is between 15 and 30 inclusive, more generally between 18 and 25 inclusive, yet more generally between 19 and 24 inclusive, and most generally between 19 and 21 nucleotides in length, inclusive. In some embodiments, the dsRNA is between 15 and 20 nucleotides in length, inclusive, and in other embodiments, the dsRNA is between 25 and 30 nucleotides in length, inclusive. As the ordinarily skilled person will recognize, the targeted region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an mRNA molecule. Where relevant, a “part” of an mRNA target is a contiguous sequence of an mRNA target of sufficient length to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway). dsRNAs having duplexes as short as 9 base pairs can, under some circumstances, mediate RNAi-directed RNA cleavage. Most often a target will be at least 15 nucleotides in length, preferably 15-30 nucleotides in length.

In yet another embodiment, the RNA of an iRNA, e.g., a dsRNA, is chemically modified to enhance stability or other beneficial characteristics. The nucleic acids featured in the invention may be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Modifications include, for example, (a) end modifications, e.g., 5′ end modifications (phosphorylation, conjugation, inverted linkages, etc.) 3′ end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), (b) base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases, (c) sugar modifications (e.g., at the 2′ position or 4′ position) or replacement of the sugar, as well as (d) backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of RNA compounds useful in the embodiments described herein include, but are not limited to RNAs containing modified backbones or no natural internucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In particular embodiments, the modified RNA will have a phosphorus atom in its internucleoside backbone.

Modified RNA backbones can include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those) having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included. Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,835, 826; 6,858,715; 6,867,289; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; 7,834,171; 7,919,612; 7,960,360; 7,989,603; 8,309,707; 6,524,681; and U.S. Pat. RE39464, each of which is herein incorporated by reference

Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts. Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, each of which is herein incorporated by reference.

In other RNA mimetics suitable or contemplated for use in iRNAs, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

Some embodiments featured in the invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂— [wherein the native phosphodiester backbone is represented as —O—P—O—CH₂—] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

Modified RNAs can also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein can include one of the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modifications include O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)._(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10. In other embodiments, dsRNAs include one of the following at the 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an iRNA, or a group for improving the pharmacodynamic properties of an iRNA, and other substituents having similar properties. In some embodiments, the modification includes a 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in examples herein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples herein below.

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications can also be made at other positions on the RNA of an iRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,700,920; 8,084,600; 8,124,745; 8,377,644 each of which is herein incorporated by reference.

An iRNA can also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, also herein incorporated by reference.

The RNA of an iRNA can also be modified to include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2′ and 4′ carbons. This structure effectively “locks” the ribose in the 3′-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Representative U.S. patents that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125; and 7,399,845, each of which is herein incorporated by reference in its entirety.

Another modification of the RNA of an iRNA featured in the invention involves chemically linking to the RNA one or more ligands, moieties or conjugates that enhance the activity, cellular distribution, pharmacokinetic properties, or cellular uptake of the iRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

In some embodiments the CTC marker gene-targeted therapy can comprise an agent that binds to the CTC marker gene expression product and an agent that is chemotherapeutic. In some embodiments, the CTC marker gene-targeted therapy comprises a CTC marker gene-binding antibody reagent and a chemotherapeutic agent. A CTC marker gene-binding antibody reagent can be an antibody reagent that binds, e.g. a CTC marker gene polypeptide. The binding antibody reagent can be an inhibitor or can exhibit no inhibitory effect on its own. By binding to the CTC marker gene, and thereby a CTC, it concentrates and localizes the chemotherapeutic agent at CTC cells in the circulation and/or stroma of the tumor—increasing efficacy and reducing side effects.

In some embodiments, the CTC marker gene-targeted therapy comprises a CTC marker gene-binding antibody reagent that binds a marker gene selected from Table 14. In some embodiments, the CTC marker gene-targeted therapy comprises a CTC marker gene-binding antibody reagent that binds a marker gene selected from the group consisting of: IL6ST, SULF2, and SV2A.

As used herein the term “chemotherapeutic agent” refers to any chemical or biological agent with therapeutic usefulness in the treatment of diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms and cancer as well as diseases characterized by hyperplastic growth. These agents can function to inhibit a cellular activity upon which the cancer cell depends for continued proliferation. In some aspect of all the embodiments, a chemotherapeutic agent is a cell cycle inhibitor or a cell division inhibitor. Categories of chemotherapeutic agents that are useful in the methods of the invention include alkylating/alkaloid agents, antimetabolites, hormones or hormone analogs, and miscellaneous antineoplastic drugs. Most of these agents are directly or indirectly toxic to cancer cells. In one embodiment, a chemotherapeutic agent is a radioactive molecule. One of skill in the art can readily identify a chemotherapeutic agent of use (e.g. see Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology 2nd ed. 2000 Churchill Livingstone, Inc; Baltzer L, Berkery R (eds): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer D S, Knobf M F, Durivage H J (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993). In some embodiments, the chemotherapeutic agent can be a cytotoxic chemotherapeutic. The term “cytotoxic agent” as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (e.g. At211, I1131, I1125, Y90, Re186, Re188, Sm153, Bi212, P32 and radioactive isotopes of Lu), chemotherapeutic agents, and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof.

Non-limiting examples of chemotherapeutic agents can include gemcitabine, cisplastin, paclitaxel, carboplatin, bortezomib, AMG479, vorinostat, rituximab, temozolomide, rapamycin, ABT-737, PI-103; alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB 1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL® paclitaxel (Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE® Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE.™vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including the treatment regimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (Tykerb™); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva®)) and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above.

In some embodiments, the binding antibody reagent and the chemotherapeutic agent can be directly conjugated and/or bound to each other, e.g. an antibody-drug conjugate. In some embodiments, binding can be non-covalent, e.g., by hydrogen, electrostatic, or van der waals interactions, however, binding may also be covalent. By “conjugated” is meant the covalent linkage of at least two molecules. In some embodiments, the composition can be an antibody-drug conjugate.

In some embodiments, the binding antibody reagent can be bound to and/or conjugated to multiple chemotherapeutic molecules. In some embodiments, the ratio of a given chemotherapeutic molecule to the binding antibody reagent molecule can be from about 1:1 to about 1,000:1, e.g. a single antibody binding reagent molecule can be linked to, conjugated to, etc. from about 1 to about 1,000 individual chemotherapeutic molecules.

In some embodiments, the binding antibody reagent and the chemotherapeutic agent can be present in a scaffold material. Scaffold materials suitable for use in therapeutic compositions are known in the art and can include, but are not limited to, a nanoparticle; a matrix; a hydrogel; and a biomaterial, biocompatible, and/or biodegradable scaffold material. As used herein, the term “nanoparticle” refers to particles that are on the order of about 10⁻⁹ or one billionth of a meter. The term “nanoparticle” includes nanospheres; nanorods; nanoshells; and nanoprisms; and these nanoparticles may be part of a nanonetwork.

The term “nanoparticles” also encompasses liposomes and lipid particles having the size of a nanoparticle. As used herein, the term “matrix” refers to a 3-dimensional structure comprising the components of a composition described herein (e.g. a binding reagent, kinase inhibitor, and/or EGFR inhibitor). Non-limiting examples of matrix structures include foams; hydrogels; electrospun fibers; gels; fiber mats; sponges; 3-dimensional scaffolds; non-woven mats; woven materials; knit materials; fiber bundles; and fibers and other material formats (See, e.g. Rockwood et al. Nature Protocols 2011 6:1612-1631 and US Patent Publications 2011/0167602; 2011/0009960; 2012/0296352; and U.S. Pat. No. 8,172,901; each of which is incorporated by reference herein in its entirety). The structure of the matrix can be selected by one of skill in the art depending upon the intended application of the composition, e.g. electrospun matrices can have greater surface area than foams.

In some embodiments, the scaffold is a hydrogel. As used herein, the term “hydrogel” refers to a three-dimensional polymeric structure that is insoluble in water but which is capable of absorbing and retaining large quantities of water to form a stable, often soft and pliable, structure. In some embodiments, water can penetrate in between the polymer chains of the polymer network, subsequently causing swelling and the formation of a hydrogel. In general, hydrogels are superabsorbent. Hydrogels have many desirable properties for biomedical applications. For example, they can be made nontoxic and compatible with tissue, and they are highly permeable to water, ions, and small molecules. Hydrogels are super-absorbent (they can contain over 99% water) and can be comprised of natural (e.g., silk) or synthetic polymers, e.g., PEG.

As used herein, “biomaterial” refers to a material that is biocompatible and biodegradable. As used herein, the term “biocompatible” refers to substances that are not toxic to cells. In some embodiments, a substance is considered to be “biocompatible” if its addition to cells in vitro results in less than or equal to approximately 20% cell death. In some embodiments, a substance is considered to be “biocompatible” if its addition to cells in vivo does not induce inflammation and/or other adverse effects in vivo. As used herein, the term “biodegradable” refers to substances that are degraded under physiological conditions. In some embodiments, a biodegradable substance is a substance that is broken down by cellular machinery. In some embodiments, a biodegradable substance is a substance that is broken down by chemical processes.

In some embodiments, the methods described herein relate to treating a subject having or diagnosed as having cancer with a CTC marker-gene targeted therapy. In some embodiments, the cancer can be pancreatic cancer. Subjects having cancer can be identified by a physician using current methods of diagnosing cancer. Symptoms and/or complications of cancer, e.g. pancreatic cancer, which characterize these conditions and aid in diagnosis are well known in the art and include but are not limited to, pain in the upper abdomen, heartburn, nausea, vomiting, diarrhea, cachexia, jaundice, pulmonary embolism, Trousseau syndrome, and diabetes mellitus. Tests that may aid in a diagnosis of, e.g. pancreatic cancer include, but are not limited to, liver function tests, CA19-9 tests, CT and endoscopic ultrasound. A family history of pancreatic cancer or exposure to risk factors for pancreatic cancer (e.g. smoking or drinking) can also aid in determining if a subject is likely to have cancer or in making a diagnosis of cancer.

The compositions and methods described herein can be administered to a subject having or diagnosed as having cancer, e.g. pancreatic cancer. In some embodiments, the methods described herein comprise administering an effective amount of compositions described herein, e.g. a CTC marker-gene targeted therapy to a subject in order to alleviate a symptom of a cancer. As used herein, “alleviating a symptom of a cancer” is ameliorating any condition or symptom associated with the cancer. As compared with an equivalent untreated control, such reduction is by at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as measured by any standard technique. A variety of means for administering the compositions described herein to subjects are known to those of skill in the art. Such methods can include, but are not limited to oral, parenteral, intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, cutaneous, topical, injection, or intratumoral administration. Administration can be local or systemic.

The term “effective amount” as used herein refers to the amount of a CTC marker-gene targeted therapy needed to alleviate at least one or more symptom of the disease or disorder, and relates to a sufficient amount of pharmacological composition to provide the desired effect. The term “therapeutically effective amount” therefore refers to an amount of CTC marker-gene targeted therapy that is sufficient to provide a particular anti-cancer effect when administered to a typical subject. An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease. Thus, it is not generally practicable to specify an exact “effective amount”. However, for any given case, an appropriate “effective amount” can be determined by one of ordinary skill in the art using only routine experimentation.

Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dosage can vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxic and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50/ED50. Compositions and methods that exhibit large therapeutic indices are preferred. A therapeutically effective dose can be estimated initially from cell culture assays. Also, a dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of a CTC marker-gene targeted therapy, which achieves a half-maximal inhibition of symptoms) as determined in cell culture, or in an appropriate animal model. Levels in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by a suitable bioassay, e.g., assay for CTC levels, among others. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.

In some embodiments, the technology described herein relates to a pharmaceutical composition comprising a CTC marker-gene targeted therapy as described herein, and optionally a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents and/or dispersion media. The use of such carriers and diluents is well known in the art. Some non-limiting examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, methylcellulose, ethyl cellulose, microcrystalline cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium stearate, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; (22) C₂-C₁₂ alcohols, such as ethanol; and (23) other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservative and antioxidants can also be present in the formulation. The terms such as “excipient”, “carrier”, “pharmaceutically acceptable carrier” or the like are used interchangeably herein. In some embodiments, the carrier inhibits the degradation of the active agent, e.g. a CTC marker-gene targeted therapy as described herein.

In some embodiments, the pharmaceutical composition comprising a CTC marker-gene targeted therapy as described herein can be a parenteral dose form. Since administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or suspended in a pharmaceutically acceptable vehicle for injection, suspensions ready for injection, and emulsions. In addition, controlled-release parenteral dosage forms can be prepared for administration of a patient, including, but not limited to, DUROS®-type dosage forms and dose-dumping.

Suitable vehicles that can be used to provide parenteral dosage forms of a CTC marker-gene targeted therapy as disclosed within are well known to those skilled in the art. Examples include, without limitation: sterile water; water for injection USP; saline solution; glucose solution; aqueous vehicles such as but not limited to, sodium chloride injection, Ringer's injection, dextrose Injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible vehicles such as, but not limited to, ethyl alcohol, polyethylene glycol, and propylene glycol; and non-aqueous vehicles such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate. Compounds that alter or modify the solubility of a pharmaceutically acceptable salt of a CTC marker-gene targeted therapy as disclosed herein can also be incorporated into the parenteral dosage forms of the disclosure, including conventional and controlled-release parenteral dosage forms.

Pharmaceutical compositions comprising a CTC marker-gene targeted therapy can also be formulated to be suitable for oral administration, for example as discrete dosage forms, such as, but not limited to, tablets (including without limitation scored or coated tablets), pills, caplets, capsules, chewable tablets, powder packets, cachets, troches, wafers, aerosol sprays, or liquids, such as but not limited to, syrups, elixirs, solutions or suspensions in an aqueous liquid, a non-aqueous liquid, an oil-in-water emulsion, or a water-in-oil emulsion. Such compositions contain a predetermined amount of the pharmaceutically acceptable salt of the disclosed compounds, and may be prepared by methods of pharmacy well known to those skilled in the art. See generally, Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams, and Wilkins, Philadelphia Pa. (2005).

Conventional dosage forms generally provide rapid or immediate drug release from the formulation. Depending on the pharmacology and pharmacokinetics of the drug, use of conventional dosage forms can lead to wide fluctuations in the concentrations of the drug in a patient's blood and other tissues. These fluctuations can impact a number of parameters, such as dose frequency, onset of action, duration of efficacy, maintenance of therapeutic blood levels, toxicity, side effects, and the like. Advantageously, controlled-release formulations can be used to control a drug's onset of action, duration of action, plasma levels within the therapeutic window, and peak blood levels. In particular, controlled- or extended-release dosage forms or formulations can be used to ensure that the maximum effectiveness of a drug is achieved while minimizing potential adverse effects and safety concerns, which can occur both from under-dosing a drug (i.e., going below the minimum therapeutic levels) as well as exceeding the toxicity level for the drug. In some embodiments, the CTC marker-gene targeted therapy can be administered in a sustained release formulation.

Controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled release counterparts. Ideally, the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled-release formulations include: 1) extended activity of the drug; 2) reduced dosage frequency; 3) increased patient compliance; 4) usage of less total drug; 5) reduction in local or systemic side effects; 6) minimization of drug accumulation; 7) reduction in blood level fluctuations; 8) improvement in efficacy of treatment; 9) reduction of potentiation or loss of drug activity; and 10) improvement in speed of control of diseases or conditions. Kim, Cherng-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000).

Most controlled-release formulations are designed to initially release an amount of drug (active ingredient) that promptly produces the desired therapeutic effect, and gradually and continually release other amounts of drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug being metabolized and excreted from the body. Controlled-release of an active ingredient can be stimulated by various conditions including, but not limited to, pH, ionic strength, osmotic pressure, temperature, enzymes, water, and other physiological conditions or compounds.

A variety of known controlled- or extended-release dosage forms, formulations, and devices can be adapted for use with the salts and compositions of the disclosure. Examples include, but are not limited to, those described in U.S. Pat. Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185 B1; each of which is incorporated herein by reference. These dosage forms can be used to provide slow or controlled-release of one or more active ingredients using, for example, hydroxypropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems (such as OROS® (Alza Corporation, Mountain View, Calif. USA)), or a combination thereof to provide the desired release profile in varying proportions.

The methods described herein can further comprise administering a second agent and/or treatment to the subject, e.g. as part of a combinatorial therapy. Non-limiting examples of a second agent and/or treatment can include radiation therapy, surgery, and chemotherapeutic agents as described above herein.

In certain embodiments, an effective dose of a composition comprising a CTC marker gene-targeted therapy as described herein can be administered to a patient once. In certain embodiments, an effective dose of a composition comprising a CTC marker gene-targeted therapy can be administered to a patient repeatedly. For systemic administration, subjects can be administered a therapeutic amount of a composition comprising a CTC marker gene-targeted therapy, such as, e.g. 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, or more.

In some embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after treatment biweekly for three months, treatment can be repeated once per month, for six months or a year or longer. Treatment according to the methods described herein can reduce levels of a marker or symptom of a condition, e.g. CTC levels by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% or more.

The dosage of a composition as described herein can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment. With respect to duration and frequency of treatment, it is typical for skilled clinicians to monitor subjects in order to determine when the treatment is providing therapeutic benefit, and to determine whether to increase or decrease dosage, increase or decrease administration frequency, discontinue treatment, resume treatment, or make other alterations to the treatment regimen. The dosing schedule can vary from once a week to daily depending on a number of clinical factors, such as the subject's sensitivity to the CTC marker gene-targeted therapy. The desired dose or amount of activation can be administered at one time or divided into subdoses, e.g., 2-4 subdoses and administered over a period of time, e.g., at appropriate intervals through the day or other appropriate schedule. In some embodiments, administration can be chronic, e.g., one or more doses and/or treatments daily over a period of weeks or months. Examples of dosing and/or treatment schedules are administration daily, twice daily, three times daily or four or more times daily over a period of 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, or more. A composition comprising a CTC marker gene-targeted therapy can be administered over a period of time, such as over a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period.

The dosage ranges for the administration of a CTC marker gene-targeted therapy, according to the methods described herein depend upon, for example, the form of the CTC marker gene-targeted therapy, its potency, and the extent to which symptoms, markers, or indicators of a condition described herein are desired to be reduced, for example the percentage reduction desired for CTC levels. The dosage should not be so large as to cause adverse side effects. Generally, the dosage will vary with the age, condition, and sex of the patient and can be determined by one of skill in the art. The dosage can also be adjusted by the individual physician in the event of any complication.

The efficacy of a CTC marker gene-targeted therapy in, e.g. the treatment of a condition described herein, or to induce a response as described herein (e.g. reduction of CTC levels) can be determined by the skilled clinician. However, a treatment is considered “effective treatment,” as the term is used herein, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced e.g., by at least 10% following treatment according to the methods described herein. Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and/or the incidence of a condition treated according to the methods described herein or any other measurable parameter appropriate, e.g. tumor size and/or growth. Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization, or need for medical interventions (i.e., progression of the disease is halted). Methods of measuring these indicators are known to those of skill in the art and/or are described herein. Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human or an animal) and includes: (1) inhibiting the disease, e.g., preventing a worsening of symptoms (e.g. pain or inflammation); or (2) relieving the severity of the disease, e.g., causing regression of symptoms. An effective amount for the treatment of a disease means that amount which, when administered to a subject in need thereof, is sufficient to result in effective treatment as that term is defined herein, for that disease. Efficacy of an agent can be determined by assessing physical indicators of a condition or desired response, (e.g. CTC levels). It is well within the ability of one skilled in the art to monitor efficacy of administration and/or treatment by measuring any one of such parameters, or any combination of parameters. Efficacy can be assessed in animal models of a condition described herein, for example treatment of cancer, e.g. pancreatic cancer. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change in a marker is observed, e.g. a change in CTC levels.

For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

For convenience, certain terms employed herein, in the specification, examples and appended claims are collected here.

The terms “decrease”, “reduced”, “reduction”, or “inhibit” are all used herein to mean a decrease by a statistically significant amount. In some embodiments, “reduce,” “reduction” or “decrease” or “inhibit” typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. As used herein, “reduction” or “inhibition” does not encompass a complete inhibition or reduction as compared to a reference level. “Complete inhibition” is a 100% inhibition as compared to a reference level. A decrease can be preferably down to a level accepted as within the range of normal for an individual without a given disorder.

The terms “increased”, “increase”, “enhance”, or “activate” are all used herein to mean an increase by a statically significant amount. In some embodiments, the terms “increased”, “increase”, “enhance”, or “activate” can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a marker or symptom, a “increase” is a statistically significant increase in such level.

As used herein, a “subject” means a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. In some embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms, “individual,” “patient” and “subject” are used interchangeably herein.

Preferably, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of cancer. A subject can be male or female.

A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment (e.g. cancer) or one or more complications related to such a condition, and optionally, have already undergone treatment for cancer or the one or more complications related to cancer. Alternatively, a subject can also be one who has not been previously diagnosed as having cancer or one or more complications related to cancer. For example, a subject can be one who exhibits one or more risk factors for cancer or one or more complications related to cancer or a subject who does not exhibit risk factors.

A “subject in need” of treatment for a particular condition can be a subject having that condition, diagnosed as having that condition, or at risk of developing that condition.

As used herein, the term “cancer” or “tumor” refers to an uncontrolled growth of cells which interferes with the normal functioning of the bodily organs and systems. A subject who has a cancer or a tumor is a subject having objectively measurable cancer cells present in the subject's body. Included in this definition are benign and malignant cancers, as well as dormant tumors or micrometastases. Cancers which migrate from their original location and seed vital organs can eventually lead to the death of the subject through the functional deterioration of the affected organs.

The term “agent” refers generally to any entity which is normally not present or not present at the levels being administered to a cell, tissue or subject. An agent can be selected from a group including but not limited to: polynucleotides; polypeptides; small molecules; and antibodies or antigen-binding fragments thereof. A polynucleotide can be RNA or DNA, and can be single or double stranded, and can be selected from a group including, for example, nucleic acids and nucleic acid analogues that encode a polypeptide. A polypeptide can be, but is not limited to, a naturally-occurring polypeptide, a mutated polypeptide or a fragment thereof that retains the function of interest. Further examples of agents include, but are not limited to a nucleic acid aptamer, peptide-nucleic acid (PNA), locked nucleic acid (LNA), small organic or inorganic molecules; saccharide; oligosaccharides; polysaccharides; biological macromolecules, peptidomimetics; nucleic acid analogs and derivatives; extracts made from biological materials such as bacteria, plants, fungi, or mammalian cells or tissues and naturally occurring or synthetic compositions. An agent can be applied to the media, where it contacts the cell and induces its effects. Alternatively, an agent can be intracellular as a result of introduction of a nucleic acid sequence encoding the agent into the cell and its transcription resulting in the production of the nucleic acid and/or protein environmental stimuli within the cell. In some embodiments, the agent is any chemical, entity or moiety, including without limitation synthetic and naturally-occurring non-proteinaceous entities. In certain embodiments the agent is a small molecule having a chemical moiety selected, for example, from unsubstituted or substituted alkyl, aromatic, or heterocyclyl moieties including macrolides, leptomycins and related natural products or analogues thereof. Agents can be known to have a desired activity and/or property, or can be selected from a library of diverse compounds. As used herein, the term “small molecule” can refer to compounds that are “natural product-like,” however, the term “small molecule” is not limited to “natural product-like” compounds. Rather, a small molecule is typically characterized in that it contains several carbon-carbon bonds, and has a molecular weight more than about 50, but less than about 5000 Daltons (5 kD). Preferably the small molecule has a molecular weight of less than 3 kD, still more preferably less than 2 kD, and most preferably less than 1 kD. In some cases it is preferred that a small molecule have a molecular mass equal to or less than 700 Daltons.

Aptamers are short synthetic single-stranded oligonucleotides that specifically bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells and tissues. These small nucleic acid molecules can form secondary and tertiary structures capable of specifically binding proteins or other cellular targets, and are essentially a chemical equivalent of antibodies. Aptamers are highly specific, relatively small in size, and non-immunogenic. Aptamers are generally selected from a biopanning method known as SELEX (Systematic Evolution of Ligands by Exponential enrichment) (Ellington et al. Nature. 1990; 346(6287):818-822; Tuerk et al., Science. 1990; 249(4968):505-510; Ni et al., Curr Med Chem. 2011; 18(27):4206-14; which are incorporated by reference herein in their entireties). Methods of generating an apatmer for any given target are well known in the art. Preclinical studies using, e.g. aptamer-siRNA chimeras and aptamer targeted nanoparticle therapeutics have been very successful in mouse models of cancer and HIV (Ni et al., Curr Med Chem. 2011; 18(27):4206-14).

As used herein, the terms “protein” and “polypeptide” are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxy groups of adjacent residues. The terms “protein”, and “polypeptide” refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogs, regardless of its size or function. “Protein” and “polypeptide” are often used in reference to relatively large polypeptides, whereas the term “peptide” is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms “protein” and “polypeptide” are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.

As used herein, the term “nucleic acid” or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analog thereof. The nucleic acid can be either single-stranded or double-stranded. A single-stranded nucleic acid can be one nucleic acid strand of a denatured double-stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double-stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable nucleic acid molecules are DNA, including genomic DNA or cDNA. Other suitable nucleic acid molecules are RNA, including mRNA.

As used herein, the terms “treat,” “treatment,” “treating,” or “amelioration” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder, e.g. cancer. The term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with a cancer. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a disease is reduced or halted. That is, “treatment” includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and/or decreased mortality, whether detectable or undetectable. The term “treatment” of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).

As used herein, the term “pharmaceutical composition” refers to the active agent in combination with a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry. The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

As used herein, the term “administering,” refers to the placement of a compound as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject.

The term “statistically significant” or “significantly” refers to statistical significance and generally means a two standard deviation (2SD) or greater difference.

Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean ±1%.

As used herein the term “comprising” or “comprises” is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the method or composition, yet open to the inclusion of unspecified elements, whether essential or not.

The term “consisting of” refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

As used herein the term “consisting essentially of” refers to those elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment.

The singular terms “a,” “an,” and “the” include plural referents unless context clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, “e.g.” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.”

Definitions of common terms in cell biology and molecular biology can be found in “The Merck Manual of Diagnosis and Therapy”, 19th Edition, published by Merck Research Laboratories, 2006 (ISBN 0-911910-19-0); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); Benjamin Lewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN-10: 0763766321); Kendrew et al. (eds.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8) and Current Protocols in Protein Sciences 2009, Wiley Intersciences, Coligan et al., eds.

Unless otherwise stated, the present invention was performed using standard procedures, as described, for example in Sambrook et al., Molecular Cloning: A Laboratory Manual (4 ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (1995); or Methods in Enzymology: Guide to Molecular Cloning Techniques Vol. 152, S. L. Berger and A. R. Kimmel Eds., Academic Press Inc., San Diego, USA (1987); Current Protocols in Protein Science (CPPS) (John E. Coligan, et. al., ed., John Wiley and Sons, Inc.), Current Protocols in Cell Biology (CPCB) (Juan S. Bonifacino et. al. ed., John Wiley and Sons, Inc.), and Culture of Animal Cells: A Manual of Basic Technique by R. Ian Freshney, Publisher: Wiley-Liss; 5th edition (2005), Animal Cell Culture Methods (Methods in Cell Biology, Vol. 57, Jennie P. Mather and David Barnes editors, Academic Press, 1st edition, 1998) which are all incorporated by reference herein in their entireties.

Other terms are defined herein within the description of the various aspects of the invention.

All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

The technology described herein is further illustrated by the following examples which in no way should be construed as being further limiting.

Some embodiments of the technology described herein can be defined according to any of the following numbered paragraphs:

-   -   1. A method of detecting circulating tumor cells (CTCs) in a         sample, the method comprising:         -   measuring the level of a PC-CTC marker gene expression             product in the sample; and determining that PC-CTCs are             present if the detected level of the marker gene expression             product is greater than a reference level.     -   2. The method of paragraph 1, wherein the CTCs are pancreatic         cancer CTCs.     -   3. The method of any of paragraphs 1-2, wherein the method         further comprises a first step of isolating the CTCs from the         sample.     -   4. The method of any of paragraphs 1-3, wherein the expression         product is a nucleic acid.     -   5. The method of paragraph 4, wherein the level of the         expression product is determined using a method selected from         the group consisting of:         -   RT-PCR; quantitative RT-PCR; Northern blot; microarray based             expression analysis; next-generation sequencing; and RNA in             situ hybridization.     -   6. The method of any of paragraphs 1-3, wherein the expression         product is a polypeptide.     -   7. The method of paragraph 6, wherein the level of the         expression product is determined using a method selected from         the group consisting of:         -   Western blot; immunoprecipitation; enzyme-linked             immunosorbent assay (ELISA); radioimmunological assay (RIA);             sandwich assay; fluorescence in situ hybridization (FISH);             immunohistological staining; radioimmunometric assay;             immunofluoresence assay; mass spectroscopy; FACS; and             immunoelectrophoresis assay.     -   8. The method of any of paragraphs 1-7, wherein the CTC marker         gene is selected from Table 7; Table 8; or Table 14.     -   9. The method of any of paragraphs 1-8, wherein the CTC marker         gene is selected from the group consisting of:         -   ABI3BP; ADAMTS5; ADAMTSL1; ANG; ARSA; C1RL; C3; C4A; C4B;             CCDC80; CD109; CHI3L1; CLEC3B; CMTM3; CMTM7; COL14A1;             COL1A2; COL3A1; COL4A6; CSF1; DAG1; DCN; DMKN; FBLN1; FGF1;             FMOD; GPC3; GPC4; HMGB1; IFNAR2; IGFBP5; IL16; LAMA4; LTBP4;             MFAP1A; NID2; OGN; PDAP1; PF4; PLAT; PODN; PRELP; RSPO1;             SERPING1; SLURP1; SOD3; SPARC; SPOCK2; SPON2; SULF1; SULF2;             TGFB2; TGM2; THBD; THBS1; THSD4; TIMP2; TNXB; TPT1; TWSG1             and WNT4.     -   10. The method of any of paragraphs 1-8, wherein the CTC marker         gene is selected from the group consisting of:         -   ALDH1A1; ALDH1A2; IGFBP5; KLF4; DCN; SPARC; WNT; TGFB2;             VEGF; COL1A2; COL3A1; and TIMP2.     -   11. The method of any of paragraphs 1-9, wherein the CTC marker         gene is selected from the group consisting of:         -   ALDH1A2; IGFBP5; KLF4; DCN; and SPARC.     -   12. The method of any of paragraphs 1-9, wherein the CTC marker         gene is selected from the group consisting of:         -   ALDH1A2; IGFBP5; KLF4; and DCN.     -   13. The method of any of paragraphs 1-9, wherein the CTC marker         gene is selected from the group consisting of:         -   TPT1; HMGB1; SPON 2; SPARC; and ARSA.     -   14. The method of any of paragraphs 1-9, wherein the CTC marker         gene is selected from the group consisting of:         -   IL6ST; ARSA; TIMP2; CD55; SULF2; ITGA6; SDC4; CDON; and             SV2A.     -   15. A method of treating cancer in a subject, the method         comprising administering a therapeutically effective amount of a         CTC marker gene-targeted therapy to the subject.     -   16. The method of paragraph 15, wherein the cancer is pancreatic         cancer.     -   17. The method of any of paragraphs 15-16, wherein the CTC         marker gene-targeted therapy comprises an inhibitor of a CTC         marker gene.     -   18. The method of paragraph 17, wherein the inhibitor is an         antibody reagent.     -   19. The method of paragraph 17, wherein the inhibitor is an         inhibitory nucleic acid reagent.     -   20. The method of any of paragraphs 15-19, wherein the CTC         marker gene-targeted therapy comprises a CTC marker gene-binding         antibody reagent and a chemotherapeutic agent.     -   21. The method of any of paragraphs 15-20, wherein the subject         is a subject determined to have an elevated level of CTCs and/or         an elevated level of a CTC marker gene present in the blood         and/or stroma of the cancer.     -   22. The method of any of paragraphs 15-21, wherein the CTC         marker gene-targeted therapy comprises a CTC marker gene-binding         antibody reagent that binds a marker gene selected from the         group consisting of:         -   IL6ST, SULF2, and SV2A.     -   23. A method of determining if a subject is likely to respond to         treatment with a CTC marker gene-targeted therapy, the method         comprising:         -   measuring the level of a CTC marker gene expression product             present in the blood and/or stroma of a cancer; and         -   determining that the subject is likely to respond to the             treatment if the level of the expression product is             increased relative to a reference level.     -   24. The method of paragraph 23, wherein the method further         comprises a first step of isolating the CTCs from the sample.     -   25. The method of any of paragraphs 23-24, wherein the cancer is         pancreatic cancer.     -   26. The method of any of paragraphs 23-25, wherein the         expression product is a nucleic acid.     -   27. The method of paragraph 26, wherein the level of the         expression product is determined using a method selected from         the group consisting of:         -   RT-PCR; quantitative RT-PCR; Northern blot; microarray based             expression analysis; next-generation sequencing; and RNA in             situ hybridization.     -   28. The method of any of paragraphs 23-26, wherein the         expression product is a polypeptide.     -   29. The method of paragraph 28, wherein the level of the         expression product is determined using a method selected from         the group consisting of:         -   Western blot; immunoprecipitation; enzyme-linked             immunosorbent assay (ELISA); radioimmunological assay (RIA);             sandwich assay; fluorescence in situ hybridization (FISH);             immunohistological staining; radioimmunometric assay;             immunofluoresence assay; mass spectroscopy; FACS; and             immunoelectrophoresis assay.     -   30. The method of any of paragraphs 23-29, wherein the PC-CTC         marker gene is selected from Table 7; Table 8; or Table 14.     -   31. The method of any of paragraphs 23-30, wherein the CTC         marker gene is selected from the group consisting of:         -   ABI3BP; ADAMTS5; ADAMTSL1; ANG; ARSA; C1RL; C3; C4A; C4B;             CCDC80; CD109; CHI3L1; CLEC3B; CMTM3; CMTM7; COL14A1;             COL1A2; COL3A1; COL4A6; CSF1; DAG1; DCN; DMKN; FBLN1; FGF1;             FMOD; GPC3; GPC4; HMGB1; IFNAR2; IGFBP5; IL16; LAMA4; LTBP4;             MFAP1A; NID2; OGN; PDAP1; PF4; PLAT; PODN; PRELP; RSPO1;             SERPING1; SLURP1; SOD3; SPARC; SPOCK2; SPON2; SULF1; SULF2;             TGFB2; TGM2; THBD; THBS1; THSD4; TIMP2; TNXB; TPT1; TWSG1             and WNT4.     -   32. The method of any of paragraphs 23-31, wherein the CTC         marker gene is selected from the group consisting of:         -   ALDH1A1; ALDH1A2; IGFBP5; KLF4; DCN; SPARC; WNT; TGFB2;             VEGF; COL1A2; COL3A1; and TIMP2.     -   33. The method of any of paragraphs 23-31, wherein the CTC         marker gene is selected from the group consisting of:         -   ALDH1A2; IGFBP5; KLF4; DCN; and SPARC.     -   34. The method of any of paragraphs 23-31, wherein the CTC         marker gene is selected from the group consisting of:         -   ALDH1A2; IGFBP5; KLF4; and DCN.     -   35. The method of any of paragraphs 23-31, wherein the CTC         marker gene is selected from the group consisting of:         -   TPT1; HMGB1; SPON 2; SPARC; and ARSA.     -   36. The method of any of paragraphs 23-31, wherein the CTC         marker gene is selected from the group consisting of:         -   IL6ST; ARSA; TIMP2; CD55; SULF2; ITGA6; SDC4; CDON; and             SV2A.     -   37. A method of monitoring the treatment of a subject, the         method comprising:         -   administering a cancer therapy to a subject in need thereof;         -   measuring the level of a CTC marker gene expression product             present in the blood and/or stroma of a cancer; and         -   determining that the subject is responding if the level of             the CTC marker gene expression product is decreased relative             to the reference level and determining that the subject is             not responding to the treatment if the CTC marker gene             expression product is not decreased relative to the             reference level.     -   38. The method of paragraph 37, wherein the cancer is pancreatic         cancer.     -   39. The method of any of paragraphs 37-38, wherein the reference         level is the level of the gene expression product in the patient         prior to the administering step.     -   40. The method of any of paragraphs 37-39, wherein the method         further comprises a first step of isolating the CTCs from the         sample.     -   41. The method of any of paragraphs 37-40, wherein the         expression product is a nucleic acid.     -   42. The method of paragraph 41, wherein the level of the         expression product is determined using a method selected from         the group consisting of:         -   RT-PCR; quantitative RT-PCR; Northern blot; microarray based             expression analysis; next-generation sequencing; and RNA in             situ hybridization.     -   43. The method of any of paragraphs 37-40, wherein the         expression product is a polypeptide.     -   44. The method of paragraph 43, wherein the level of the         expression product is determined using a method selected from         the group consisting of:         -   Western blot; immunoprecipitation; enzyme-linked             immunosorbent assay (ELISA); radioimmunological assay (RIA);             sandwich assay; fluorescence in situ hybridization (FISH);             immunohistological staining; radioimmunometric assay;             immunofluoresence assay; mass spectroscopy; FACS; and             immunoelectrophoresis assay.     -   45. The method of any of paragraphs 37-44, wherein the PC-CTC         marker gene is selected from Table 7; Table 8; or Table 14.     -   46. The method of any of paragraphs 37-45, wherein the CTC         marker gene is selected from the group consisting of:         -   ABI3BP; ADAMTS5; ADAMTSL1; ANG; ARSA; C1RL; C3; C4A; C4B;             CCDC80; CD109; CHI3L1; CLEC3B; CMTM3; CMTM7; COL14A1;             COL1A2; COL3A1; COL4A6; CSF1; DAG1; DCN; DMKN; FBLN1; FGF1;             FMOD; GPC3; GPC4; HMGB1; IFNAR2; IGFBP5; IL16; LAMA4; LTBP4;             MFAP1A; NID2; OGN; PDAP1; PF4; PLAT; PODN; PRELP; RSPO1;             SERPING1; SLURP1; SOD3; SPARC; SPOCK2; SPON2; SULF1; SULF2;             TGFB2; TGM2; THBD; THBS1; THSD4; TIMP2; TNXB; TPT1; TWSG1             and WNT4.     -   47. The method of any of paragraphs 37-46, wherein the CTC         marker gene is selected from the group consisting of:         -   ALDH1A1; ALDH1A2; IGFBP5; KLF4; DCN; SPARC; WNT; TGFB2;             VEGF; COL1A2; COL3A1; and TIMP2.     -   48. The method of any of paragraphs 37-46, wherein the CTC         marker gene is selected from the group consisting of:         -   ALDH1A2; IGFBP5; KLF4; DCN; and SPARC.     -   49. The method of any of paragraphs 37-46, wherein the CTC         marker gene is selected from the group consisting of:         -   ALDH1A2; IGFBP5; KLF4; and DCN.     -   50. The method of any of paragraphs 37-46, wherein the CTC         marker gene is selected from the group consisting of:         -   TPT1; HMGB1; SPON 2; SPARC; and ARSA.     -   51. The method of any of paragraphs 37-46, wherein the CTC         marker gene is selected from the group consisting of:         -   IL6ST; ARSA; TIMP2; CD55; SULF2; ITGA6; SDC4; CDON; and             SV2A.

EXAMPLES Example 1 Single Cell RNA-Sequencing of Mouse Pancreatic Circulating Tumor Cells Reveals their Expression of ECM Proteins

Circulating Tumor Cells (CTCs) are shed from primary tumors into the bloodstream, mediating the hematogenous spread of cancer to distant organs. Using a pancreatic cancer mouse model, a microfluidic device was applied to isolate CTCs independently of tumor epitopes, subjecting these to single cell RNA-sequencing. CTCs clustered into multiple subsets, distinct from primary tumors and cancer cell lines. While proliferative signatures were generally low, CTCs were enriched for MAPK, as well as WNT, TGF-β, Neurotrophin, Toll-like receptor, and B-cell receptor signaling pathways. CTCs were highly enriched for expression of the stem-cell associated gene Aldh1a2. Their virtually universal expression of Igfbp5 and Klf4 was correlated with a subset of primary tumor cells localized to the epithelial/stromal boundary, consistent with the presence of both epithelial and mesenchymal markers in CTCs. The very high CTC expression of stromal-derived extracellular matrix proteins, including Dcn and Sparc, indicates microenvironmental contributions to metastasis and identifies unexpected therapeutic targets.

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer deaths in the US, with a 6% overall survival at 5 years (Society, 2013). The high mortality of this cancer stems from the rapid dissemination of tumor cells leading to widespread metastasis. While local tissue and lymphatic invasion are evident even in early PDAC, the presence of circulating tumor cells (CTCs) in the bloodstream ultimately leads to spread of cancer to distant organs. CTCs are rare, estimated at one to ten tumor cells among ten billion normal blood cells in a milliliter of blood. As such, their isolation and molecular analysis has posed a significant technological challenge (Pantel et al., 2008; Yu et al., 2011). Given their role in blood-borne metastasis, CTC populations are likely to be enriched for metastatic precursors, and their analysis may identify potential therapeutic targets, as well as providing opportunities for early detection of pancreatic cancer.

Genetically engineered mouse pancreatic cancer models have provided important insight into the progression of this disease. Specifically, the genetically engineered LSL-Kras^(G12D), Trp53^(flox/flox or +), Pdx1-Cre (KPC) mouse model recapitulates the histological progression from preneoplastic pancreatic intraepithelial neoplasia (PanlN) lesions to invasive carcinoma (Bardeesy et al., 2006). Recent studies have suggested that epithelial-to-mesenchymal transition (EMT) occurs early in this model potentially enhancing tumor invasiveness (Rhim et al., 2012). In an initial molecular characterization of mouse pancreatic CTCs, RNA sequencing of CTC-enriched populations was performed, thereby identifying activation of non-canonical WNT signaling as a recurrent event, potentially contributing to the anoikis resistance of circulating epithelial cells (Yu et al., 2012). In that study, analysis of purified CTC populations was accomplished using single molecule RNA sequencing, combined with digital subtraction of matched leukocyte RNA reads, so as to derive a CTC-enriched expression signature. However, transcriptomic analysis of such partially purified cell populations is limited by depth of coverage to the most highly differentially expressed genes, and such studies of bulk CTC populations cannot resolve the degree of heterogeneity across these poorly understood cell populations

To achieve a deep RNA sequencing profile of CTCs at the single cell level, a novel inertial focusing-enhanced device, the CTC-iChip, which allows high efficiency negative depletion of normal blood cells, leaving unattached CTCs in solution where they can be selected and analyzed as single cells (Ozkumur et al., 2013) was used. By avoiding tumor epitope-specific capture, such as targeting the epithelial marker EpCAM, the CTC-iChip is unbiased in isolating cancer cells with both epithelial and mesenchymal characteristics. Further, the high quality of RNA purified from viable, untagged CTCs is particularly well suited for detailed transcriptomic analysis. Finally, the use of a mouse model of pancreatic cancer allows for simultaneous analysis of primary tumor and CTCs, while the shared driver mutations across different animals facilitates the identification of CTC-specific heterogeneity. Described herein is a comprehensive transcriptome analysis of CTCs at the single cell level, pointing to distinct cell subsets within CTC populations, signaling pathways that are enriched in CTCs, and identifying unique CTC markers and therapeutic targets.

Results

Isolation of Mouse Pancreatic CTCs.

The CTC-iChip, an integrated microfluidic cell separation platform applied directly to whole blood specimens for isolation of CTCs (Ozkumur et al., 2013) was used in the experiments described herein. It combines initial hydrodynamic size-based separation of all nucleated cells (leukocytes (WBC) and CTCs) away from red blood cells, platelets and plasma, with subsequent inertial focusing of the nucleated cells within a single streamline to achieve high efficiency in-line magnetic sorting. While tumor epitopes are highly variable, WBC cell surface markers are well established; applying magnetic-conjugated anti-WBC antibodies to this very high throughput microfluidic cell separation device can thus exclude the vast majority of WBCs to reveal a small number of untagged CTCs (FIG. 1A). The CTC-iChip was adapted for depletion of murine hematopoietic cells and applied to the KPC pancreatic cancer mouse model. This PDAC model generates significant numbers of CTCs (Rhim et al., 2012; Yu et al., 2012). Whole blood labeling using 100 anti-CD45 beads per WBC achieved >10³ depletion in normal mice, mice bearing orthotopic tumors, and the genetically engineered KPC mice (FIGS. 1B and 4A-4C).

CTC recovery was measured as a mean of 95% (+/−3% std), using GFP-tagged NB508 mouse pancreatic cancer cells spiked into whole mouse blood and processed through the CTC-iChip (FIGS. 4A-4C). NB508 cells were previously generated from a pancreatic tumor arising in the same Kras/Trp53-driven KPC mouse model (Bardeesy et al., 2006). In comparison, only 35% recovery of the same cells was achieved using an alternative microfluidic platform based on anti-EpCAM capture of mouse CTCs (Yu et al., 2012). Applying the CTC-iChip to orthotopic tumors derived from pancreatic inoculation of GFP-tagged NB508 cells generated >1000 CTCs/mL in all three mice tested (FIGS. 4A-4C). Finally, testing the CTC-iChip with the genetically engineered KPC model, followed by dual immunofluorescence staining of isolated cells for the epithelial marker pan-cytokeratin (CK) versus the leukocyte marker CD45, revealed a median 118 CTCs/mL (mean 429 CTCs/mL; range 0-1694) (FIG. 1C). No CK positive cells were isolated from 7 healthy control mice. The vast majority of CD45 positive cells that failed to be deflected in the microfluidic device retained some immunomagnetic beads on their surface. Thus, CTCs were readily distinguished from WBCs in the CTC-iChip product, enabling single cell manipulation without requiring staining for epithelial-specific cell surface epitopes, such as EpCAM.

Single CTC RNA-Sequencing.

Five tumor-bearing KPC mice generated a total of 168 single CTCs that were subjected to a modified initial cDNA amplification and library protocol (Tang et al., 2010), and screened for RNA quality (Gapdh, Actb), presence of pancreatic markers (Krt8, Krt18, Krt19, Pdx1), and absence of WBC markers (Cd45/Ptprc) (FIGS. 5A-5C). Of these, 75 (45%) were of sufficient quality to proceed to further amplification and library construction for next generation sequencing. It is noteworthy that a majority of candidate CTCs (55%) appeared morphologically intact but had degraded RNA. These cells likely represent tumor cells that have lost viability in the bloodstream. Given the rapid processing of blood samples from mouse models, the minimal shear condition in the microfluidic device, and the preserved RNA quality of control cells processed identically, it is unlikely that cells underwent such damage during in vitro purification. For comparison with pancreatic CTCs, single cell RNA-sequencing was also performed on 12 WBCs from a control mouse, 12 mouse embryonic fibroblasts (MEFs), and 16 single cells from the mouse NB508 pancreatic cancer cell line. Over 90% of single cells from NB508 and MEF cultures met criteria for sequencing quality, highlighting the high frequency of CTCs with compromised RNA templates under the same conditions. To compare CTC profiles to that of matched parental tumors harvested at the time of CTC isolation, bulk RNA from each primary tumor was diluted to 1 or 10 cell equivalents (10 or 100 pg RNA) and subjected to the same amplification and RNA-sequencing protocol (n=34; min 8 replicates from 4 matched tumors).

Single cell RNA sequencing performance was comparable for all samples analyzed, with a mean 4.4-8.5 million reads, of which a mean 46-61% were uniquely aligned to the genome (FIGS. 5A-5C). Genome aligned reads were annotated and counted using UCSC Known Gene transcriptome reference and normalized in reads per million (RPM). Normalized reads were then analyzed by unsupervised hierarchical clustering (data not shown). Single cell transcriptomes from MEFs, the NB508 pancreatic cancer cell line and normal WBCs were tightly clustered, supporting the analytic reliability of the RNA sequencing strategy. Five distinct clusters of candidate CTCs were identified, all of which were distinct from matched primary tumor sequences, as well as from cancer-derived cell lines. Principal component analysis demonstrates the clustering and inter-relationships of these different groups (FIG. 2).

The uniform genetic drivers of PDAC in the KPC mouse model made it possible to quantify measures of cellular heterogeneity in CTCs derived from individual mice and across different mice. Single cell heterogeneity within each CTC cluster was assessed by calculating the intra-cluster correlation coefficients, where lower correlation coefficients reflect higher heterogeneity (FIGS. 5A-5C). As expected, CTC clusters showed considerably more heterogeneity (mean 0.42, 95% CI 0.36-0.47) than single cells derived from the NB508 cancer cell line (mean 0.86, 95% CI 0.80-0.91, p-value 1.2×10⁻¹⁵). To assess heterogeneity of cells within a primary PDAC, a conditional Tomato/EGFP (mT/mG) expression marker (Muzumdar et al., 2007) was crossed with the KPC mouse to generate a lineage-tagged mouse tumor (KPC-mT/mG), which could be used to isolate individual EGFP positive primary tumor cells away from contaminating stromal cells. A primary tumor (TuGMP3) was disaggregated into single cell suspension and 20 EGFP positive cells were subjected to RNA sequencing. The single primary tumor cells clustered well within the previously analyzed bulk tumor material (data not shown), with a heterogeneity score (mean 0.38, 95% CI 0.28-0.47) similar to that of CTCs (p-value 0.49).

In summary, described herein is the single cell RNA-sequencing of mouse pancreatic CTCs isolated without positive selection bias, along with parental tumors, an established genotype-matched cancer cell line, MEFs and WBCs. CTCs clustered separately from the primary tumor (both bulk tumor and isolated single cells) and from the tumor-derived cell line, with comparable degrees of intercellular heterogeneity between CTCs and primary tumor cells.

Defining Subsets of Pancreatic CTCs.

To identify and classify candidate CTCs, gene sets for known epithelial, hematopoietic, and endothelial markers were applied across all clustered samples. As expected, epithelial markers (Krt7, Krt8, Krt18, Krt19, Epcam, Egfr, Cdh1) were highly expressed in primary pancreatic tumors and in the cancer cell line NB508, and nearly absent in the non-epithelial MEFs and in normal WBCs (data not shown). In contrast, hematopoietic markers (Ptprc/Cd45, Csf3r/Cd114, Cd14, Fcgr3/Cd16, Itga2b/Cd41, Itgb3/Cd61) were present in normal WBCs, and absent in NB508 and MEFs. Some expression of hematopoietic markers was detectable in the bulk primary tumor samples, consistent with varying degrees of leukocytic infiltrates. No specific cluster of endothelial cells was identified, based on expression of characteristic markers (Cdh5/Cd144, Vwf Thbd/Cd141, Pecaml/Cd31, Mcam/Cd146, Sele/E-selectin, Cd34) and absence of epithelial and hematopoietic markers.

Interrogation of single cells isolated by CD45-depletion from tumor-bearing mice, using the epithelial, hematopoietic and endothelial markers, revealed five major candidate CTC groupings (Clusters 1, 3, 4, 5 and 9; data not shown). Clusters 3, 4, and 5 were all part of a larger grouping, showing strong expression of epithelial markers, consistent with “classical” CTCs (denoted CTC-c). A subset of these cells expressed Cd34, an endothelial progenitor marker that is also found in mesenchymal cells including MEFs (data not shown) and stromal cells (Krause et al., 1994), but other characteristic endothelial lineage markers were absent. Clusters 1 and 9 were more complex, with the former noteworthy for enrichment of platelet markers CD41 (Itga2b) and CD61 (Itgb3) (hence denoted CTC-plt), and the latter having a prominent cellular proliferation signature (CTC-pro).

To better define the characteristics of each candidate CTC cluster, a non-parametric differential gene expression analysis including a rank product (RP) methodology adapted to variations in absolute transcript levels and differences in transcriptome representation from cell to cell was used (Breitling et al., 2004). Setting very stringent parameters (FDR≦0.01), the control comparison of primary tumors versus WBCs identified 927 genes relatively overexpressed in tumors and 293 genes high in WBCs, including the expected differential expression of epithelial tumor markers keratin 7, 8, 18, and 19, versus the leukocyte specific CD45 (data not shown). Comparing the “classical” CTC-c cluster to WBCs also showed enrichment for cytokeratin 18 and 19 in CTCs versus CD45 in WBCs, validating the RP methodology to identify relevant differentially expressed genes between single cell populations.

The most abundant CTC cluster, CTC-c, comprised 41 of 75 cells (55%) meeting established criteria for epithelial tumor cells (versus CTC-plt: 32%; CTC-pro: 13%). Of note, the only mouse with multiple gross metastases (MP7) had large numbers of CTCs within this class. Compared with matched primary tumors CTC-c cells had 878 transcripts increased in expression and 774 genes with reduced expression (Table 2). Gene Ontology (GO) analysis of CTC-c enriched genes (Table 3) indicated enrichment for signatures associated with cellular interactions with environmental signals (GO:0045785—positive regulation of cell adhesion; GO:0048584—positive regulation of response to stimulus), cell shape and structure (GO: 0030036—actin cytoskeleton organization; GO:0060429—epithelium development), and transcriptional states (GO:0045449—regulation of transcription; GO:0051276—chromosome organization). To evaluate the contribution of signaling pathways activated by external stimuli in CTC-c cells, the enriched genes were annotated using the KEGG database (Table 1). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis similarly showed enrichment for focal adhesion (odds ratio [OR]2.7, q-value 6.7 3 10.4) and regulation of actin cytoskeleton (OR 2.4, q-value 0.005). Notably, of the KEGG signaling pathways annotated, the mitogen-activated protein kinase (MAPK) pathway was most highly enriched Most highly represented was the MAPK pathway (OR 2.2, q-value 0.006); MAPK signaling is already activated in the Kras^(G12D) driven primary tumor. However, while MSigDB Kras dependency signatures were enriched in primary tumors compared with CTCs, the latter had increased expression of Braf, Mras and Rras2, pointing to alternative paths to further activate MAPK in CTCs. This finding is consistent with another study that identified the MAPK pathway as being the most highly enriched in pancreatic CTCs using microarray based methodologies (Sergeant et al., 2012).

CTC enriched genes also had representation of well established signaling pathways involved with metastasis, including TGF-β (Ikushima and Miyazono, 2010; Siegel and Massague, 2003), WNT (Anastas and Moon, 2013; Clevers and Nusse, 2012; Katoh and Katoh, 2007), and VEGF (Carmeliet and Jain, 2011; Folkman, 1995). In this cohort of pancreatic cancer CTCs, Wnt4 and Tgfb2 were most highly enriched in CTCs relative to primary tumor, implicating autocrine signaling involving these major pathways. In addition to these well defined contributors to metastasis, CTC expression analyses also revealed activation of unexpected signaling pathways, including the neurotrophin, toll-like receptor, and B-cell receptor pathways. Neurotrophin pathway activation has been reported in pancreatic cancer, particularly in association with increased perineural invasion (Miknyoczki et al., 1996; Miknyoczki et al., 1999; Ohta et al., 1997; Wang et al., 2009; Zhang et al., 2005). Toll-like receptor and B-cell receptor pathways had less representation among CTC reads, but they suggest aberrant activation of immunomodulatory signaling components. Ultimately, the establishment of CTC-derived cultures will be required to test the functional significance of these activated signaling pathways.

While single cells within the CTC-c cluster fulfilled characteristic criteria for tumor cells, defining the identity of the non-classical CTC clusters, CTC-plt and CTC-pro, required additional analyses. Compared with CTC-c, single cells within the CTC-plt cluster had a high enrichment for wound healing and hemostasis signatures, as well as MSigDB platelet and megakaryocyte expression profiles (Table 4). This indicates that these cells are either circulating megakaryocytes/giant platelets or CTCs covered with adherent platelets. Tumor cell specific lineage tagging supports the identification of CTC-plt cells being of tumor origin. Eighteen EGFP lineage-tagged single CTCs from two KPC-mT/mG mice were subjected to single cell RNA sequencing: a total of 9 CTCs from the two mice (7/7 CTCs from mouse GMP1 and 2/11 from mouse GMP2) were included within CTC-plt, using unsupervised hierarchical clustering (data not shown). Thus, the CTC-plt cluster includes CTCs that exhibit strong platelet markers, most likely derived from transcripts encoded by adherent platelets. Interestingly, CTC-plt cells maintained their distinct segregation from CTC-c even after digital removal of all annotated platelet transcripts (data not shown). It is therefore possible that the adherence of abundant platelets may modulate the intrinsic CTC expression profile, as recently suggested by in vitro modeling experiments (Labelle et al., 2011).

The CTC-pro cluster was most similar to both the NB508 pancreatic cancer cell line and MEFs, and it was enriched for the cellular proliferation marker Mki67 when compared to CTC-c. Multiple lineages are likely to have contributed to this complex grouping: CTCs from KPC mice with tumor-restricted, lineage-tagged EGFP expression clustered with CTC-pro (data not shown), noteworthy for abundant expression of Mki67 and an annotated cell cycle signature in MSigDB (Whitfield et al., 2002) (data not shown). One single cell within the CTC-pro cluster was derived from the pancreatic cancer cell line NB508, while another (MP3-2) had high keratin/high E-cadherin expression characteristic of classical CTCs (data not shown). Nonetheless, another sub-cluster contained immune and dendritic cells, identified by their expression of antigen processing and presentation genes (GO:0019886—antigen processing and presentation of exogenous peptide antigen via MHC class II; Table 5). Taken together, the CTC-pro cluster appears to represent a grouping of highly proliferative cells, of which a subset are tumor-derived.

Together, unbiased isolation and RNA sequencing evaluation of single pancreatic CTCs indicate that over half of these are nonviable with RNA at various stages of degradation. Among the remaining viable CTCs, three major classes are distinguishable by unsupervised clustering: the classical subset (CTC-c) accounts for 55%, with a second platelet adherent group (CTC-plt; 32%) and a third heterogeneous cluster marked by proliferative signatures (CTC-pro; 13%). Given their most clearly defined tumor-derived characteristics, we selected the CTC-c cluster for detailed analysis of metastasis-associated pathways.

Pancreatic CTCs Co-Express Epithelial, Mesenchymal, and Stem Cell Markers.

The relevance of EMT to early metastasis in pancreatic cancer has been supported by lineage tracing studies in the KPC mouse model (Rhim et al., 2012). In human breast cancer CTCs, a distribution of epithelial and mesenchymal markers within individual CTCs was recently reported by the inventors, reflecting both tumor histology and response or resistance to diverse therapies (Yu et al., 2013). To directly test for EMT in the mouse pancreatic CTCs, established epithelial (E) and mesenchymal (M) markers (Kalluri and Weinberg, 2009) were used to evaluate each cell within the CTC-c cluster (data not shown). Compared with the primary tumor, CTC-c cells demonstrated clear loss of the epithelial markers E-cadherin (Cdh1) and Muc1, whereas mesenchymal transcripts were mixed, with some showing increased expression (Cdh11, Vim) and others with reduced levels (S100a4, Itga5, Sdc1) (FIGS. 3A and 3B). Notably, even the mesenchymal genes that were upregulated in CTCs showed a high degree of heterogeneous expression across single cells (data not shown). In contrast, loss of epithelial marks, including E-cadherin (Cdh1) was nearly universal across all classical CTCs.

CTCs are also thought to be enriched for metastatic precursors, capable of initiating metastatic tumor deposits. The relationship between such precursor cells and postulated cancer stem cells is uncertain, as is the relevance of established stem cell markers in identifying these cells. Proposed pancreatic cancer stem cell genes (Rasheed and Matsui, 2012; Rasheed et al., 2010) were evaluated in the single cell RNA sequencing reads (FIG. 3B). Among all candidate markers tested (Aldh1a1, Aldh1a2, Prom1/Cd133, Cd44, Met, EpCAM), only Aldh1a1 and Aldh1a2 were enriched in CTCs. Classical CTCs expressed predominantly the Aldh1a2 isoform, while CTC-plt cells were enriched for Aldh1a1, but these isoforms were also co-expressed within some single CTCs. MEFs, NB508 pancreatic cancer cells and normal WBCs also expressed Aldh1a1, but not Aldh1a2 (data not shown). Within single CTCs, there was no correlation between expression of Aldh1 isoforms and enrichment for the mesenchymal genes Cdh11 or Vim, suggesting that these two biomarkers are not intrinsically linked.

Given the identification of Aldh1a2 as a potential stem-like marker expressed by CTCs, its expression within matched primary tumors was tested using RNA in situ hybridization (RNA-ISH). Expression patterns within tumors were heterogeneous: Aldh1a2 expressing cells were primarily localized within the “stromal” or non-epithelial (i.e. keratin low) compartment of the tumor (data not shown). The origin of these non-epithelial cells, which are particularly abundant in pancreatic cancer, is likely to be mixed. Both histological evaluation and negative KRAS mutational analysis (Biankin et al., 2012; Ogino et al., 2005) in human pancreatic cancer have indicated that most of these cells represent reactive fibroblasts or stroma, rather than being of tumor origin. However, lineage tracing in KPC mice has recently shown that a small fraction of these supposedly stromal cells are in fact tumor-derived, presumably having undergone EMT to appear fibroblastic (Rhim et al., 2012). Interestingly, the mouse with the most metastases and the highest number of Aldh1a2 positive CTCs, MP7, also had the primary tumor with the highest levels of Aldh1a2. In that case, Aldh1a2-positive cells were present diffusely in the stromal compartment, as well as comprising a small subpopulation of the epithelial (keratin high) component (data not shown). Thus, classical CTCs, which are keratin-high, express the stem cell-associated gene Aldh1a2, whose expression in primary tumors is restricted to the stromal (keratin low) compartment and only a small subpopulation of epithelial cells.

Classical CTCs Share Expression of Stromal Enriched Genes.

Beside the evident diversity of CTCs, shared transcripts were sought that might provide further insight into their cell of origin within the primary tumor, the mechanisms by which they invade and survive within the bloodstream, and ultimately identify potential CTC-specific therapeutic targets. Rigorous criteria were selected to identify the most highly enriched CTC transcripts (RP score<300), expressed at very high levels (>100 RPM) in ≧90% of all classical CTCs. Three genes met these criteria: Decorin (Dcn), a extracellular matrix proteoglycan expressed in tumor stroma across a variety of different cancers (Adany et al., 1990; Bostrom et al., 2013; Henke et al., 2012; Hunzelmann et al., 1995; Iozzo and Cohen, 1994; Mu et al., 2013; Nash et al., 2002); Insulin-like growth factor binding protein 5 (Igfbp5), an extracellular growth factor binding protein expressed in human PDAC reported to have both pro and anti-proliferative properties (Johnson et al., 2006; Johnson and Haun, 2009); and Kruppel-like factor 4 (Klf4), one of the key stem cell (iPS) reprogramming factors (Takahashi and Yamanaka, 2006), which has been implicated in pancreatic cancer development (Brembeck and Rustgi, 2000; Prasad et al., 2005; Wei et al., 2010). By RNA-ISH, Dcn was expressed diffusely in the stromal elements of the tumor (FIG. 6). Remarkably, both Igfbp5 and Klf4 were expressed focally, predominantly within stromal-appearing cells that border the epithelial compartments of the tumor (data not shown). RNA-ISH of EGFP lineage restricted primary tumors confirmed that the Igfbp5 positive cells at the epithelial/stromal interface are of tumor origin (data not shown). In addition to this transitional region, analysis of Klf4 in this EGFP-tagged tumor also found expression in a subset of epithelial ducts (data not shown). Of note, while they are expressed in only a small subset of primary tumor cells, both Igfbp5 and Klf4 are highly co-expressed in 85% of all classical CTCs. Together with the mixed epithelial/mesenchymal markers evident in CTCs, these observations raise the possibility that many CTCs are derived from foci at the epithelial/stromal interface, that may be defined by Igfbp5 and Klf4 expression.

In addition to the three most highly expressed transcripts, CTCs were noteworthy for high level expression of genes implicated in stromal cell matrix. Gene ontology analysis of all CTC-enriched genes (Table 3) identified 60 extracellular proteins (GO:0044421, OR 1.7, q-value 6.4×10⁻³), of which 32 are found in proteinaceous extracellular matrix (ECM) (GO:0005578, OR 2.4, q-value 4.8×10⁻³). Recent studies have highlighted the importance of the reactive stroma to pancreatic cancer pathogenesis and metastasis (Feig et al., 2012; Neesse et al., 2013; Neesse et al., 2011; Olive et al., 2009; Provenzano et al., 2012), however, the expression of these stroma-associated ECM genes within tumor cells in circulation was unexpected. To identify the predominant stromal enriched genes in the mouse pancreatic tumor model, we performed RP differential expression analysis between the bulk tumor samples representing tumor cells mixed with reactive stromal cells versus purified EGFP-tagged single cells from the primary tumor (TuGMP3). A total of 51 proteinaceous ECM genes were enriched in bulk tumors versus single primary tumor cells (GO:0005578, OR 4.8, q-value 3.4×10⁻¹⁸). Of these, 6 genes (Ccdc80, Col1a2, Col3a1, Dcn, Sparc, Timp2) were shared with the previously identified CTC-enriched gene set (data not shown). Decorin (Dcn), as noted above, was identified as the most highly enriched (median 10,686 rpm) in CTCs with high level expression (>100 rpm) in 98% of CTCs. The second most abundant gene was Sparc (median 3,913 rpm) with high expression in 88% of CTCs. These two genes were co-expressed at high levels in 88% of classical CTCs. RNA-ISH of primary tumors for both Dcn (FIG. 6) and Sparc (data not shown) confirmed that these genes are expressed throughout the reactive stroma and are not present in the epithelial keratin-rich regions of primary tumors.

The expression of stromal-derived ECM genes is a common feature of all classical CTCs, yet a mouse-specific bias in distribution among these genes was evident, despite their identical Kras/p53 genetic drivers. This mouse-specific clustering was evident in the unsupervised analysis (p-value<2.2×10⁻¹⁶). For instance, sub-cluster 3 was over-represented with single CTCs from mouse MP6, while sub-cluster 4 was enriched for mouse MP7, and sub-cluster 5 for mouse MP2. Of 68 transcripts differentially expressed between the CTCs of mice MP2 and MP7 by RP analysis, gene ontology indicated significant enrichment for 11 extracellular proteins (GO:0044421, OR 3.8, q-value 0.06), 7 of which are found in proteinaceous ECM (GO:0005578, OR 6.3, q-value 0.05) (data not shown). Together, these data indicate that most CTCs derived from a mouse pancreatic cancer model express at high levels a set of ECM genes normally found in the stromal, rather than the epithelial compartment of the primary tumor. This may reflect the origin of many CTCs at the epithelial/stromal interface, consistent with their expression of uniquely restricted markers such as Igfbp5 and Klf4. The fact that individual genetically matched mouse tumors generate CTCs with both shared and unique patterns of ECM gene expression suggests tumor-specific invasion pathways that are superimposed upon fundamental characteristics of CTCs. The high levels of extracellular proteins expressed by CTCs provide unexpected opportunities for targeting these metastatic precursors.

Human Pancreatic CTCs Express the ECM Protein SPARC.

To determine the relevance of ECM protein expression to human disease, CTCs were isolated from the blood of metastatic PDAC patients and subjected to single cell RNA-sequencing. Analysis of 7 pancreatic CTCs from 3 patients revealed that the majority expressed keratins defining their epithelial origins and a total of 13 of 60 extracellular protein genes enriched in mouse CTCs were expressed at high levels (>100 rpm) in at least one human pancreatic CTC (FIG. 7). Human SPARC was the only gene found at high levels in all human pancreatic CTCs. Analysis of human prostate and breast CTCs also show significant expression of extracellular proteins including SPARC highlighting that these targets are commonly shared in metastatic epithelial cancer cells (data not shown). RNA-ISH of Sparc/SPARC in both mouse and human PDAC found expression confined primarily to the stromal compartment of tumors (data not shown). SPARC expression was found in 196/198 (99%) human primary PDAC tumors and 36% of positive tumors had some detectable SPARC in epithelial tumor cells albeit the minority of the overall signal. The presence of SPARC as an extracellular protein permits antibody directed therapies that target SPARC. Together these data indicate that findings in mouse pancreatic CTCs can be found in human disease and offer both novel biomarkers and therapeutic targets.

Discussion

Described herein is a detailed analysis of CTC composition and diversity, using single cell RNA sequencing. In total, high quality transcriptomes were achieved in 93 single mouse pancreatic CTCs, which were compared with 20 single cells from matched primary tumors, as well as bulk tumor preparations, and with 16 cells from an immortalized cell line established from the same mouse pancreatic tumor model. The use of a mouse model, which closely matches human PDAC, made it possible to compare primary tumor specimens isolated simultaneously with the CTCs. Given the shared Kras/Trp53 genetic drivers in the KPC mouse model, it was also possible to examine CTC heterogeneity within individual mice and across different animals. Finally, the use of the CTC-iChip technology enabled the selection of untagged CTCs, irrespective of their cell surface epitopes, thus avoiding any bias associated with tumor marker-specific cell purification. Together, these observations include the following: 1. CTCs cluster into multiple subsets, including a major “classical CTC” group, and others that are marked by platelet-derived markers or proliferative signatures; 2. While individual mouse tumors may produce CTCs that fit into each of these clusters, there are unique patterns to CTCs derived from individual mice, despite their shared genetic drivers; 3. Common markers shared by virtually all classical CTCs include both epithelial and mesenchymal markers, the Aldh1a2 stem cell marker, and two highly expressed transcripts (Igfbp5 and Klf4) that identify foci localized to the epithelial/stromal boundary of primary tumors; and 4. The most highly enriched CTC-specific transcripts shared by almost all classical CTCs encode extracellular matrix proteins associated with the tumor stromal compartment.

Compared with previous RNA sequencing of partially purified, bulk CTC populations, the single cell analysis reported here provides considerably more depth of tumor cell-specific reads. As such, the detailed analysis of classical CTCs from the mouse pancreatic cancer model is unprecedented. It is demonstrated herein that pancreatic cancer CTCs uniformly lose expression of the epithelial marker E-cadherin (Cdh1), a key feature of epithelial-to-mesenchymal transition. However, the cells do not lose expression of other epithelial markers, such as cytokeratins, nor is there a consistent increase in classical EMT mesenchymal markers such as vimentin. As such, most classical CTCs appear arrested in a biphenotypic state. Despite their expression of cytokeratins (present in the epithelial components of the primary tumor), most other highly expressed markers in CTCs were shared with the non-epithelial or “stromal” component of the primary tumor. Among these stromal genes expressed in classical CTCs is Aldh1a2, a putative pancreatic cancer stem cell marker (Rasheed and Matsui, 2012; Rasheed et al., 2010). Whether Aldh1a2 is a functionally significant marker of cellular plasticity in metastatic precursors remains to be determined.

A provocative observation relating to the shared epithelial and mesenchymal state of classical CTCs is their virtually uniform (>85%) high level co-expression of Igfbp5 and Klf4, two genes that are only expressed in a small subpopulation of cells at the epithelial/stromal interface within primary tumors. This raises the intriguing possibility that this critical location within the tumor generates a disproportionate fraction of viable CTCs. Indeed, tumor cells that are actively undergoing EMT are presumably enriched at the epithelial-stromal function, contributing to the mixed lineage of the tumor stroma, with both tumor-derived and non-malignant reactive cell types. The potential roles of both IGF signaling and Klf4 transcriptional regulation in embryonic development and pancreatic malignancy make their unique expression pattern in both tumors and CTCs particularly noteworthy.

Finally, the most unexpected observation from this single CTC RNA sequencing study is the very high level abundance of ECM proteins on the vast majority of classical CTCs. Notably, prior evaluation of matched primary and metastatic breast tumors identified the most prevalent gene expression difference as enrichment for ECM molecules in the metastases, comprising some 18% of differentially expressed genes (Weigelt et al., 2005). While this has been interpreted as reflecting differences in the local environment of the metastatic site, the present data indicate that ECM proteins are highly expressed by CTCs themselves. By analogy with the classical “seed versus soil” debate (Fidler, 2003), CTCs may in fact be seeds carrying some of their own soil.

The ultimate goal of detailed molecular analysis of CTCs is to understand the process by which they are generated and their therapeutic vulnerabilities. In this regard, an important observation derived from the present single CTC RNA sequencing analysis is the unexpected expression of extracellular proteins with a preponderance of proteins found in ECM. Two of the most abundant and commonly shared ECM proteins in CTCs are Dcn and Sparc, both of which are established tumor stromal genes. Notably, Sparc expressing stroma appears to bind albumin-conjugated chemotherapy-containing nanoparticles (nab-paclitaxel) allowing for increased cytotoxicity and efficacy in human PDAC (Neuzillet et al., 2013; Von Hoff et al., 2011; Yardley, 2013). Indeed, considerable effort has been directed to targeting pancreatic cancer stroma as a means of improving delivery of chemotherapeutics and stripping tumor cells of their supportive microenvironment (Neesse et al., 2011; Olive et al., 2009; Provenzano et al., 2012; Rasheed et al., 2012). The finding that these gene products are also expressed by CTCs indicates that antibody-directed therapies can be used not only against primary tumor stroma, but also to target tumor cells as they transit in the blood.

As described herein, the present CTC analyses to extend from matching them to known tumor-defining markers to interrogating them for unique properties that distinguish them from most primary tumor cells and may underlie their ability to survive in the bloodstream and generate distant metastases. Such insights into the cellular process of human cancer metastasis are critical to the goal of ultimately preventing the spread of a primary tumor to distant organs.

Experimental Procedures

Mice and cell lines. Mice with pancreatic cancer used in these experiments express Cre driven by Pdx1, LSL-Kras^(G12D), and Trp53^(lox/+) or Trp53^(lox/lox) as previously described (Bardeesy et al., 2006). EGFP pancreatic lineage tagged KPC mice were generated by breeding the mT/mG mouse (Jackson Laboratory—Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J) into the breeder pairs used for KPC mouse generation. Normal FVB mice were purchased from Jackson Laboratory. All mice care and procedures were done under MGH SRAC approved protocols.

Adaptation of CTC Enrichment Technology.

Given the desire for an unbiased enrichment system, the previously presented negative depletion technology was selected for this application (Ozkumur et al., 2013). All processing protocols were identical to those previously identified, except a rat anti-mouse CD45 antibody (BAM114, R&D Systems, USA) was conjugated to MyOne beads.

Single Cell Micromanipulation, Amplification, and Sequencing.

After whole blood anti-CD45 negative depletion, the product containing enriched cells was collected in a 35 mm petri dish and viewed using a Nikon Eclipse Ti™ inverted fluorescent microscope. Cells of interest were identified based on intact cellular morphology and lack of labeling with anti-CD45 magnetic beads. These target cells were individually micromanipulated with a 10 μm transfer tip on an Eppendorf TransferMan® NK 2 micromanipulator and ejected into PCR tubes containing RNA protective lysis buffer and immediately flash frozen in liquid nitrogen. Single cells were amplified with a modified protocol (Tang et al., 2010) and sequenced on the ABI 5500XL™ system.

RNA In Situ Hybridization (RNA-ISH).

RNA-ISH was performed according to the Affymetrix QuantiGene ViewRNA ISH Tissue-2 Plex Assay™.

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TABLE 1 Annotation of CTC enriched genes in KEGG defined signaling pathways. *indicates gene found in multiple pathway gene sets. TGF- Toll-Like Neurotropin beta Receptor VEGF MAPK Pathway WNT Pathway Pathway Pathway Pathway Pathway 1500003o03rik* Jund 1500003o03rik* Akt2* Amhr2 Akt2* 1500003o03rik* Akt2* Map3k3* Crebbp* Braf* Crebbp* Fos* Akt2* B230120h23rik Mapk1* Csnk1a1 Calm1 Dcn Ifnar2 Hspb1* Braf* Mapkapk3* Jun* Calm2 Id1 Irak4* Kdr Dusp1 Mef2c Nkd1 Irak4* Id2 Irf7 Mapk1* Dusp14 Mras Ppp3ca* Irs2 Mapk1* Jun* Mapkapk3* Dusp3 Nf1 Rock1* Jun* Rock1* Mapk1* Pla2g4a* Fas Nfkb2 Rock2* Maged1 Rock2* Nfkbia* Ppp3ca* Fgf1 Nr4a1 Siah1a Map3k3* Smad4* Tirap Src Flnc Pla2g4a* Smad4* Mapk1* Tgfb2* Tlr2 Fos* Ppp3ca* Tbl1x Nfkbia* Tgfbr2* Gadd45b Rras2 Tcf7l1 Shc1 Thbs1 Hspa2 Tgfb2* Wnt4 Ywhaz Hspb1* Tgfbr2* Jun*

TABLE 2 Significantly Expressed Genes by Rank Product (FDR < 0.01) CTC-c vs Primary Primary Tumor vs CTC-plt vs CTC-pro vs Count Tumor Enriched Gene CTC-c Enriched Gene CTC-c CTC-c 1 Upk3b Tff2 Clec1b kg:uc007pge.1 2 Ier2 Wfdc2 AU023871 kg:uc007pgd.1 3 Egr1 Lamb3 Alox12 kg:uc007pgf.1 4 Nkain4 Lad1 Itga2b kg:uc007pgg.1 5 Igfbp5 Dmbt1 Ppbp Igj 6 Slc6a4 Npy Gng11 kg:uc012enb.1 7 Klf4 Pmepa1 Vwf 2010001M09Rik 8 Tmem221 Kcnn4 Pf4 kg:uc009cfw.1 9 Arl4d Serinc2 Fcer1g kg:uc007pgi.1 10 Lrrn4 5730559C18Rik Tmem40 kg:uc007pgh.1 11 Cldn15 Muc1 Hba-a2 kg:uc007yos.1 12 Gpm6a Chi3l3 Stom Coro1a 13 Atf3 Pglyrp1 Beta-s Pou2af1 14 Ptma Arl4c Plek kg:uc011yvj.1 15 Slc9a3r1 Spp1 Srgn Glipr1 16 Fos Col15a1 Myl9 Cd52 17 Tmem119 C1qb Cd84 Cd79b 18 Ptgis Tnnt2 F5 Sec11c 19 Dcn Gkn3 Treml1 Tnfrsf17 20 Gbp2 Onecut2 Hbb-b1 Krr1 21 Dmkn Mmp7 Itgb3 Gmfg 22 Sdc4 Cd74 Gp9 Ccr9 23 Ildi2 Ctss Mpl Pycard 24 Akap2 Lamc2 Ctla2a Derl3 25 Gfpt2 Olfml3 Tubb1 Rac2 26 Klf6 Lgals4 Mylk Srgn 27 Btg2 Lcn2 F13a1 Cytip 28 Myl7 Ly6a Slamf1 Edem2 29 Igfbp6 Pak1 Rgs10 Itgb7 30 Gpr133 Capn5 Mkrn1 Lsp1 31 Oasl2 Ptprn Laptm5 Lcp1 32 Pfn1 Reg3b 1810058I24Rik Cyfip2 33 Cap1 Fmnl3 Itgb2 Nans 34 Nfkbia Sdc1 Slc2a3 Slamf7 35 Malat1 Prom1 Pcmt1 Ell2 36 Rarres2 Ankrd50 Gp5 H2-Eb1 37 Rspo1 Ccl6 Ube2o Creld2 38 Espn Slc4a11 5430417L22Rik Cd74 39 Klf9 Oraov1 Ptpn18 Blnk 40 Zbtb7c Aldh1l1 Lat Fmnl1 41 Brd2 Slc20a1 Fermt3 Snrnp70 42 Olfr1033 Cldn7 Nrgn Sec61b 43 Wt1 Acsbg1 Mrvi1 Edem1 44 Esam Las1l Lyz2 Tspan13 45 kg:uc009igb.1 C1qc Epb4.1 Psmb8 46 Tmem151a Lama5 Rasgrp2 Pim1 47 Mgll Mgat4a Treml2 Sept1 48 Csrnp1 Cldn2 Hist1h4i Cd48 49 Cd9 Mcpt2 March2 Sub1 50 Gjb5 Fxyd3 Ltbp1 Lims1 51 Lrrc61 Il4ra Nptn Ncoa2 52 Wasf2 Itga5 Abtb1 Ctnnbl1 53 Pdpn Porcn Ctla2b Fdps 54 kg:uc009ogv.1 Mast3 Prkab2 Ube2j1 55 Sdpr Scara3 Arhgdib Mettl1 56 Gpr64 Atox1 Alas2 Lax1 57 Flnc Arrdc1 Odc1 Rilpl2 58 Add3 Mmp2 Ptpn11 Ctse 59 Gata6 Saa3 Dhcr24 Glrx 60 Wfdc1 Serpinf1 Mfsd2b Fut8 61 A130040M12Rik Sox11 Gp1bb AI662270 62 Ankrd12 Prpsap1 Rbpms2 Gramd3 63 Adamtsl1 Mcpt1 Fyb Il2rg 64 C2 Mfge8 Smox Rasgrp3 65 Prss23 Col18a1 P2rx1 Impdh1 66 Ube2v1 Lyz2 Otud7b Plek 67 Cryab C1qa kg:uc007ttx.1 Ints5 68 Pkhd1l1 Acp5 Samd14 Blmh 69 Rtn1 Angptl4 Clca1 Dnmt1 70 Birc6 Ccnd1 kg:uc007tty.1 Galk1 71 Xdh Asl Gpr56 kg:uc007hxv.1 72 Cd34 Ctxn1 Sh3bgrl2 Ccdc88b 73 Rab6b Pgs1 Pttg1ip Selplg 74 Dusp1 Anapc2 Nomo1 Sar1b 75 Clic4 Cp Gnaz Lat2 76 C3 Gpx3 Mmrn1 Slc16a6 77 Rhob Lama3 Gp1ba Mki67 78 Mir3064 Rbp1 Sh3bgrl3 Dnajc3 79 Thbd Cotl1 Slc24a3 H2-Ab1 80 Dpysl2 Nek6 Sord Ndufs6 81 Cob1 Cpxm1 Nfe2 Actr3 82 Npr1 Sfrp1 Tuba4a Etnk1 83 Dnajb9 Ttr Zyx Herpud1 84 Arhgap29 Gsto1 Cnn2 Ptpn7 85 Cav1 Npepl1 Itgb5 Ctss 86 Gbp7 Usmg5 Gata1 Cs 87 Hes1 Polr2l Hist1h1c Fbxw7 88 Gm16897 Sphk1 Tbxas1 Ppp2r5c 89 Ppp1r12a Asxl1 Ptplad2 Znrd1 90 Sv2a Ctsh Bpgm Rfc2 91 Ang Egfl7 Pdlim7 Preb 92 Aldh1a2 C1qtnf6 Mmd Fcer1g 93 Cryl1 Rras G6b Dnajb11 94 Kank1 Lgi4 kg:uc009duo.1 Slc35b1 95 2210403K04Rik Hmga2 Lyz1 Sin3b 96 kg:uc009okn.1 Cep250 Tacc1 Nktr 97 Osr1 B4galt3 Dap 98 kg:uc008ewj.2 Tmem223 Mast2 99 kg:uc009tuw.1 Ltbp2 Atp2a3 100 Gadd45b Tnfrsf23 Snca 101 Ablim3 Col7a1 Stx11 102 Clec3b Ggct C030046I01Rik 103 Usp25 Rab25 Trpt1 104 Sntb2 Nedd8 Tsc22d1 105 Rock2 9430023L20Rik Prkar2b 106 Col14a1 Arl2 Cd9 107 Cd200 Wbp1 Pgm2l1 108 kg:uc008ehr.1 H2-Ab1 Gp6 109 Atp2b1 Preb Pde5a 110 Exoc4 Sgsm3 Itga6 111 Abcb1b Sfn Itga1 112 Nrgn Prrx2 Edem1 113 kg:uc009cvm.1 Ptprk Isg20 114 Ncoa4 Reg1 Cdc42ep5 115 Ndufa4 Sdcbp2 Nipal3 116 Upk1b Pcbd1 Ccdc92 117 Jun Slc25a1 Sort1 118 Syne2 Vamp5 Ly6g6c 119 kg:uc007bvx.1 Crlf1 Ubash3b 120 Ap4e1 Avil Inf2 121 Spock2 2700094K13Rik Asap1 122 Efemp1 Ctse Sec11c 123 Prpf40a Penk Gas2l1 124 Tspan5 Tmc4 Parvb 125 Lgals7 Dhrs3 Tmsb4x 126 Kif5b Ap1s1 kg:uc007xrw.1 127 Psip1 Arl6ip4 Nudt3 128 kg:uc008oki.1 9430008C03Rik Bcl2l1 129 1810014B01Rik Fcer1g B230312A22Rik 130 Ptges3 Uqcr11 Cnp 131 Limch1 Nhp2 Plp1 132 Bicd1 Plbd2 Cnst 133 Rdx Capg Rgs18 134 Pcdh15 Pnpla6 Lsm12 135 Foxn3 Ppdpf Alox5ap 136 Morf4l2 Hgfac Ppif 137 Ppp1r15a Apoe Spnb1 138 Cdc42ep3 Fam40a Ormdl3 139 Pard3b Lyz1 Hpse 140 Bicc1 2200002D01Rik Srxn1 141 Amhr2 Laptm5 2010002N04Rik 142 Gucy1a3 Qars Hist1h2bc 143 Psmb2 Tmx2 Cyba 144 Mapkapk3 Fkbp4 Chst12 145 Ube2l6 Plin2 kg:uc009sps.1 146 kg:uc007pff.1 Fcgr3 Max 147 kg:uc007ctp.1 Gkn1 Was 148 Nedd4 Snhg1 Isca1 149 Plxna4 Lsp1 Pdzk1ip1 150 2010107G12Rik Gm20605 Lyn 151 Ifhgr1 Ly6c1 Mob3a 152 Bcam Aim1 H2-T24 153 Ccnl1 2310007B03Rik Slc44a1 154 Hoxa5 Tgfbi Derl1 155 Fhl1 Tsta3 Gclm 156 1810041L15Rik Pafah1b3 Fech 157 2900002K06Rik Chid1 Ywhah 158 Hspb1 Smox Igtp 159 Podn 1500012F01Rik Myl6 160 Fam63b Tspan4 Thbs1 161 Hsp90b1 Agrn Tln1 162 Dpp4 Cfp kg:uc009apq.1 163 Gas1 Cdh1 Bcap31 164 kg:uc007zak.1 Rasgrf1 Ilk 165 Zc3h13 Nxf1 Epha1 166 Sox6 Pdrg1 2810453I06Rik 167 Arid4a Polr2j Rnf19b 168 Tnxb Suds3 Gsn 169 Tsix D0H4S114 Flna 170 Scd1 Ccl9 Arrb1 171 Jund Neat1 kg:uc007pum.1 172 Crls1 Ccdc12 Mbnl1 173 1110003E01Rik Prr24 Ccnd3 174 Rnase4 Impdh1 Pdlim1 175 Arhgef12 Card10 Ctse 176 Irf7 Cpsf1 Tspan17 177 Bbx Sema4g Gpx4 178 Sema5a Hes6 Bnip3l 179 Mau2 C130074G19Rik P2ry12 180 Abi3bp Ctrb1 kg:uc009vev.1 181 Dag1 Rnaseh2a Prkab1 182 Cyp2s1 Golm1 F2rl2 183 Sfrs18 Ctsz Stk4 184 Hspb8 Cyb561 Fhl1 185 Cnot6l Ndufs8 Rnf10 186 Twsg1 Atp6ap1 Rasa3 187 Gpc3 Srd5a1 Taldo1 188 Lrrn4cl Carkd Bysl 189 Cdh3 Cd24a Esd 190 Cyr61 Eng Aldh2 191 Cyp2d22 Tcirg1 Rhog 192 Hist1h1c Slc9a3r2 kg:uc009ecr.1 193 Aplp1 0910001L09Rik Cald1 194 Tbl1x Cox5b Wbp2 195 Pcm1 Adipor2 Ptprj 196 Ifi204 Scarf2 Tpm4 197 Nfix Myo7a Mxi1 198 Flrt2 Ppap2c Ly6g6f 199 Heg1 Pea15a Sla 200 Il6ra Sh3pxd2b Slpi 201 Ralbp1 H19 Bicd2 202 Rhoj Tpd52 Clu 203 Ktn1 2610203C20Rik Mtmr14 204 Arl6ip5 Naa10 Abca7 205 Crebbp Fermt1 Ppp1r18 206 Ppig Sap30l Kif2a 207 Akap13 Bgn Prdx6 208 Rab7 Timm13 kg:uc009ize.1 209 Plxdc2 Krt20 Calm3 210 Aldh1a1 Itga3 Dhrs1 211 Bnc2 Pfkl Cfl1 212 Slc4a4 Agpat6 Glipr2 213 Tbx18 Mrpl11 Slc25a37 214 Zbtb16 Ramp1 Atox1 215 Arid4b Hmga1 BC057079 216 Enpp2 Gpx2 Pla2g16 217 Ptplad2 0610012G03Rik Rnf144b 218 Akr1b3 9130017N09Rik Stk16 219 Gm6644 Cygb Rsad2 220 Arf5 Tmprss4 Paip2 221 Chi3l1 Paox Capzb 222 Gpr116 Endod1 Ppp1r12c 223 Cd82 Cndp2 4930412F15Rik 224 Srrm1 Suv39h1 Ninj1 225 Fmo2 Cog4 2510009E07Rik 226 Tgfb1i1 Trim27 kg:uc007vsr.1 227 Qrich1 Cyhr1 Pygb 228 Nfia Trmt1 Tlk1 229 Pmp22 Zfyve19 Myct1 230 Cdh11 Esrp1 Rnasek 231 Arid5b kg:uc008oow.1 Ctsd 232 Rbm3 Dync1h1 0610010K14Rik 233 Prelp Tab1 Bcas3 234 kg:uc007qse.1 Pla2g6 Atpif1 235 Ddx3x Timp1 Serf2 236 Sulf1 Eif3f Becn1 237 Spnb2 Abhd11 Tspan9 238 Tspan31 Pmm2 Acer2 239 Prr13 Tyrobp Vdac3 240 Ppp1cb Farsb kg:uc008kbg.1 241 Fbln1 Plod3 Oaz2 242 Gm6548 Abtb1 Serpine2 243 Uap1 Brf1 Ccdc90a 244 Mpdz Tnk2 Ndufa1 245 Sat1 Rfc2 Tssc1 246 Stim1 Stxbp2 Mboat7 247 Mll3 Pdlim7 Cd44 248 Slurp1 A430105I19Rik Cxx1c 249 Cd81 Vill Ecm1 250 Emp2 Bmp1 Mff 251 Trpm7 Mpzl1 Ptpn12 252 Crym Thy1 Mgmt 253 Enpp4 Stab1 Cox4i1 254 Raly Aldh16a1 Tollip 255 Celf2 Eif4ebp3 Cds2 256 Ap3s1 Itpripl2 Ybx1 257 C1s Mrpl52 Gypc 258 Frmd4b 2310002L13Rik Dgkd 259 Nr4a1 Mcm6 Pecam1 260 Acini Kcnk1 Ftl2 261 Plod2 Pmf1 Nt5c3 262 Id1 Cuta 1700037H04Rik 263 Creg1 Nt5dc2 Cd151 264 Zfp318 Rmnd5b Lpin2 265 Tmem140 Araf 6430548M08Rik 266 Mras Wwp2 Pon2 267 Vwa5a Lamb1 Ndufa3 268 Esyt3 Kcne3 6330578E17Rik 269 Hexb Uqcrq Mfap31 270 Nckap1 Gps1 Mink1 271 Nipal3 Rexo4 Ston2 272 Ubxn4 Coro1c Rac2 273 Zfp36 Hras1 Fyn 274 Hnrnpl Spint1 Serinc3 275 C1ra Cblc Maged2 276 Nnmt Fhod1 Ap2m1 277 Mut Atp13a1 Pacsin2 278 kg:uc008jup.1 Man2c1 Ftl1 279 Pnrc1 Vsig2 Adipor1 280 Usp8 Bpgm kg:uc009qdo.1 281 Pgcp Bap1 Snap23 282 Junb Smpd2 Tagln2 283 C1rl Ubqln4 Cox6c 284 Slc6a6 Sirt7 Creg1 285 kg:uc008znh.1 Krt23 Bsg 286 Aqp1 D8Ertd738e Cmtm6 287 Myh10 Mapk13 Cntd1 288 Slc43a3 kg:uc008bcq.1 Plekho2 289 Spint2 Polr2g Arrb2 290 Hnrnph1 Ndufs2 Pard3b 291 Arhgap28 Dad1 Mlec 292 Cfh Wnt7b Taf10 293 Brd4 Fam20c Gabarapl2 294 Fndc1 Cxxc5 Bag1 295 Star Polr2f Galnt2 296 Nfkbiz Ltf Hk1 297 Arsb 2210407C18Rik Fbxo9 298 Rnd3 Cdipt kg:uc009izd.1 299 Stard5 Glrx5 Pnpo 300 Thbs1 Gemin7 Fam46c 301 kg:uc008wkn.1 Man1b1 Pkm 302 Slc26a3 Heatr7a Ap1b1 303 Phip Arid5a Rap1b 304 Usp2 Sumo3 Itgb1 305 Golgb1 Srm St7 306 Rock1 Plscr3 Smap1 307 Rgma 2210010C17Rik Rabgap11 308 Actg1 Fam102a Tmbim4 309 BC013529 Dlst H3f3a 310 kg:uc007zwh.1 Vps37c Frmd8 311 3110062M04Rik Ngfrap1 Nlrx1 312 Cast Pold4 Oaz1 313 Mob3c Grcc10 Fam125b 314 Slc16a1 Wnt7a Hexa 315 Fam117a 2010111I01Rik Tspo 316 Pdia3 Pxdn Dcaf12 317 Trim8 Coasy Nav1 318 kg:uc009mng.1 Dctn1 Cd24a 319 eg:245190:chr7:m Ncor2 Uqcr11 320 Sbsn Postn Wipf1 321 Serpinb6b Col4a2 F10 322 Daglb Cib1 Erlec1 323 Smarca2 Tbc1d13 Map2k3 324 Mef2c Ccnl2 Stk24 325 Prrc2c Dcakd Ldlrap1 326 BC005537 Cdc34 Ehd4 327 Hsp90ab1 Atp6v0b Atp6v1f 328 Snrnp70 Abhd12 Gnas 329 Ppl Flot2 Arhgap18 330 Serpinh1 Sla2 Arhgap10 331 Sorbs3 Rhbdf1 Pitpnm1 332 Golga4 Cdh17 S100a1 333 Acbd3 Psmb5 Bin1 334 Hook3 Serf1 Ttyh3 335 Map3k3 Slc15a3 Selp 336 Rhou Sftpd Trappc9 337 Smc2 Pop5 Aes 338 C1d Nudc Taok3 339 kg:uc008dzh.1 Sh2d5 Zfand3 340 Psmd7 kg:uc007fwp.1 Stim1 341 Dab2 Mrpl37 Rnf11 342 Cep164 Rin1 Sep15 343 Crim1 Podxl kg:uc012hdk.1 344 Rtf1 Paqr5 Lgals9 345 Fxyd1 Sepx1 Cox6b1 346 H2-D1 Agr2 Riok3 347 Zfp704 Bax Slc38a10 348 Mtap1a Rxrb Rtn3 349 Ascc3 Tes B3gat2 350 Med13l Hdac6 Ccndbp1 351 Jup 1110008F13Rik Rsu1 352 Nid2 Mpnd kg:uc007upr.1 353 Kdr Gmppa Itm2b 354 Ifnar2 Gramd1a St3gal1 355 5430435G22Rik Wars Sec61g 356 Col4a6 Mtap Ptpn1 357 Il17re C1qtnf5 kg:uc012bhf.1 358 Gbp3 Mrpl28 B2m 359 Slc39a8 Mfrp Rasgrp3 360 Cfl2 Kars Memo1 361 Slc38a1 Lbp Slc39a4 362 Cuedc1 Plxnb1 Sdcbp 363 Fgf1 2700081O15Rik Tspan14 364 Gas6 Mrps24 Ubl7 365 Cldn25 Klc4 Nras 366 Sorbs1 Dctn3 Ssx2ip 367 Hspa12a Kcnq1 kg:uc007zbz.1 368 kg:uc007zts.1 Smurf1 Wbp1 369 Slc1a5 Fam162a 1110003E01Rik 370 Nr3c1 Hip1r Clip2 371 Adamts5 kg:uc007hyr.2 Gapdh 372 Gpcpd1 Gys1 Gm6578 373 Dpysl3 Sac3d1 Actn1 374 Colec12 Ndufs6 St3gal2 375 Pdcd6ip Rgl2 3110001D03Rik 376 Dst Atp5g1 Ctsz 377 Ifit4 Itgb4 kg:uc007vdl.1 378 Chst4 Sars Fam73a 379 Xist 2310003F16Rik Vcl 380 Ifi27l2a Nhp2l1 Lims1 381 Fkbp5 D19Wsu162e Lars2 382 Agap1 Cd320 Birc2 383 Ankrd11 Pigq Lamp2 384 kg:uc007qca.1 Chd3 Rasl10a 385 Syt11 Zdhhc4 Mif 386 Ptrf Eif3l Rab10 387 Krcc1 St8sia3 Pabpc1 388 Zfp488 Rcan3 Wwp2 389 Lama4 Meg3 Nqo2 390 Aebp1 Nudt4 kg:uc007fte.1 391 Fam134b Gss Plxna4 392 Tppp3 Pih1d1 Gm1821 393 Maf Limd2 Gadd45a 394 Peli1 Ap1s2 Slc25a39 395 Zfp353 BC056474 kg:uc009pet.1 396 Cdon Mms19 Ubb 397 Sarnp Clip2 Ppp1r2 398 Atxn7l3b 2310016M24Rik Rab27b 399 Pef1 Itpa Cap1 400 App Slc25a10 Jarid2 401 Mtdh Fibp Rnf11 402 Lrrc20 Higd2a Tmem50b 403 Btbd2 Snrpd2 Myh9 404 Gnb2 Eri3 Tmem128 405 Pigt Nbeal2 Stradb 406 Efna5 Trim28 Cela1 407 Tm4sf1 S100a4 Ndrg2 408 Coq10b Ivns1abp Dhrs3 409 Eif2s3x Ppp1r18 Hipk1 410 Cmah Efemp2 Atg9a 411 Sf3b1 Med22 412 Eea1 Nelf 413 Slpi 2810428I15Rik 414 Tmod3 D2Wsu81e 415 Ppp3ca Trappc6a 416 Tceal8 Trappc21 417 Anp32a Antxr2 418 Actb Rab11fip5 419 Ddx5 Ldhd 420 Cobll1 Npnt 421 Cish Acrbp 422 Nod1 Pafah1b2 423 Psd Angptl2 424 Gm10052 Fzr1 425 Lims2 Aaas 426 Stra6 Eif2b2 427 kg:uc007bgn.1 1190003J15Rik 428 Plxdc1 5730403B10Rik 429 Nfe2l1 Adamts13 430 Smpd3 Eif3b 431 Bcl10 Znrf1 432 Ilf3 Pkp3 433 Fam76a Lemd2 434 Cybrd1 Rab34 435 Gm3893 Mpv17l2 436 Siae Cdkn2b 437 Ssh2 Snrpe 438 Nfic Gm14005 439 Btf3 Prdx4 440 Sp100 Xab2 441 Ndn Dpp3 442 Matr3 Tyms 443 Gm13251 Leprotl1 444 Arhgap5 Uqcr10 445 Zbtb4 Cdk5rap3 446 Pgrmc1 Gorasp2 447 4930402H24Rik Wbp7 448 Bptf Sort1 449 Dusp3 Ddx41 450 Pla2g4a Cct3 451 Brp44l Mrps33 452 Oxct1 Frmd8 453 Stk40 1110049F12Rik 454 Ddr1 Fscn1 455 Ifi205 Ndufa2 456 Col3a1 Dpcd 457 Nipb1 Unc13a 458 Plk1s1 Eif1ad 459 Bdp1 Sgta 460 Smc3 Chaf1a 461 Ifitm3 Plxna1 462 Ndst1 Hspa9 463 Zbed6 1110014N23Rik 464 Rest Cd99l2 465 kg:uc007vnc.1 Snrpa 466 Ccdc88a Mcm7 467 Stat3 Tars2 468 Arf2 Gon4l 469 Trib1 Stk38 470 Gcap14 C1qtnf1 471 Tbc1d15 Tbrg4 472 Igf1r Tmem132a 473 Ppbp Cox6c 474 kg:uc008tky.1 Alcam 475 Rab1b Phka2 476 Krt14 Trim3 477 Med21 Ppp1r14b 478 Gja1 Gpaa1 479 Klf10 Ctps2 480 Id2 Ptpn23 481 Mfap1a Endog 482 Ogn Mrto4 483 Gpc4 Mrps6 484 Bst2 Pvr 485 Dtx2 Phgdh 486 Wac Itpr3 487 Kpna3 Polr2e 488 Kcnab1 Sec16a 489 Orai3 Mdp1 490 Gcsh Fbf1 491 Wdr92 Mcpt8 492 Olfr613 Rps6ka4 493 Tcf7l1 Mical1 494 Tgfb2 Mrpl34 495 Il16 Agpat3 496 Manf 2310044H10Rik 497 Mgst1 Myo9b 498 kg:uc008tkz.1 Ndufb10 499 Creb3l1 Apex1 500 Txndc5 Elk3 501 Klf2 Cpsf3l 502 Slu7 Tnk1 503 Ttc28 Pmvk 504 1110002B05Rik Ppp1r16a 505 Zcchc11 Arhgef5 506 Ptp4a2 Lonp1 507 Pbx1 Pla2g7 508 Clcn3 Pip5k1c 509 Tmco7 Inf2 510 Lrrc58 Pgk1 511 Eif3a Parp6 512 Cldn10 Urm1 513 H2-Q6 Mad2l2 514 Ccdc80 Ing4 515 kg:uc009iln.1 Rbck1 516 Rab5c Cant1 517 Tsc22d3 Sgpl1 518 Tm4sf5 Ehbp1l1 519 Hmgb1 Runx1 520 Sec62 Slc27a4 521 Maoa Ndufa7 522 Clec1b Mcm3ap 523 Mphosph8 1110008P14Rik 524 Oat Rassf7 525 Ncor1 Ptpmt1 526 Cyb5 Arfgap1 527 Trafd1 Sec61a1 528 Rpp25 Rps6ka1 529 kg:uc007ded.1 Ints1 530 2610101N10Rik Tpcn1 531 Il6st Iffo2 532 Evpl Trim44 533 Psmd11 kg:uc012ctw.1 534 Dync1i2 Golga2 535 Lars2 Msto1 536 Pdia4 Ppp6r3 537 Cd55 Trmt2a 538 Amfr Appl2 539 Zcchc3 Sparcl1 540 Herpud2 Rapgef1 541 Txnrd1 Zfpl1 542 Vat1 Psmc4 543 Diap1 Mosc2 544 Tmed2 Fam101b 545 Arf3 1500010J02Rik 546 Arap2 Ccdc124 547 St3gal1 Ptges 548 Man1a Fam189b 549 Rgs10 Th11 550 Tmsb4x Kctd2 551 Uba7 Olfr1372-ps1 552 C4b Hexa 553 Tmem98 Anapc5 554 Lpar2 Serpina3n 555 Gabarapl1 1810046J19Rik 556 Cmtm7 Tmem167 557 Spon2 Gm11428 558 Smarca5 Gcn1l1 559 Mxd4 Kansl3 560 Smc4 Fasn 561 Thsd4 Slc50a1 562 Gsr Smad3 563 Ptprd Trip6 564 Clip1 Atp6v1e1 565 Cln8 Chchd5 566 Rbm27 Adssl1 567 Zmat1 Nes 568 Smc6 Ap1b1 569 B2m Fcgrt 570 Irf2bp2 Ltbp3 571 Ppap2a Csf2rb 572 Zfhx4 Ssna1 573 Tob2 Mrps16 574 Rabgap1l Cyba 575 Nfkb2 Cyth2 576 Nfyc Igf2 577 Ube2d1 Pisd-ps1 578 Creb5 Atp13a2 579 Opa3 Mlph 580 Csnk1a1 Cyp4f16 581 Fam84b 2010107E04Rik 582 Ddr2 Gas5 583 Usp54 Eif3k 584 Akt2 Fam149a 585 Strn3 Mif 586 Hnrnpm B230312A22Rik 587 eg:497210:chr14:m Ppp1r12c 588 Tpt1 Tfip11 589 Naa25 Tex 10 590 Eef1a1 Slc16a3 591 Parp4 Stk16 592 Msn Epn1 593 Zbtb20 Noc4l 594 Fermt2 Rcc2 595 Bod1l Rgs12 596 Sltm Shkbp1 597 Dapk1 Got2 598 Hnrnpr Plek2 599 Baz2a Lilrb3 600 Rnf167 Ndufb5 601 Mapk1 Tesk1 602 eg:320169:chr9:p Rab24 603 4930523C07Rik Atp5j2 604 Nf1 Commd9 605 Fam53b Rtkn 606 Faim2 Prpf19 607 Tgm2 6720401G13Rik 608 Calm2 Ppa1 609 AI848100 Pgp 610 Slc10a3 Hps1 611 Ogdh Puf60 612 Arl3 Mdm2 613 Timp2 kg:uc012cgd.1 614 Atxn2 kg:uc009uim.1 615 Mll1 Pyy 616 Ces2g Zfp358 617 Mat2a Timm8b 618 Esf1 Ddx39 619 Hsp90aa1 Pgm2 620 Zfp385a kg:uc008gbp.1 621 Zfp672 Sipa1 622 Csda Mgat1 623 Pf4 Tmem208 624 Arsa Ruvbl2 625 F11r 8430410A17Rik 626 C4a Bad 627 Kpna1 Pfdn5 628 Rbbp8 Eme1 629 Oxnad1 kg:uc009mzj.1 630 Rb1cc1 Igf1 631 Setd2 Prkag1 632 Kif1b kg:uc009sua.1 633 2510002D24Rik Uap1l1 634 Cep57 Trappc4 635 Chd2 Bola2 636 Serinc5 Usp5 637 Marcksl1 Ear2 638 Shfm1 Cars 639 Bbs4 1810027O10Rik 640 Impad1 Amdhd2 641 Tbcel Phb 642 Kdelr1 Kcmf1 643 Ninl Lsmd1 644 Sytl1 Sec11c 645 Tpm3 Pcbp4 646 Rbbp6 Mepce 647 Lman1 Tpd52l2 648 Ankrd17 Trf 649 Naga Hsd17b11 650 Rbpms Pilra 651 Magt1 Atn1 652 Tfdp2 Pgf 653 Gem Nxn 654 Pde4dip Inpp5k 655 Mrgprf Actr1a 656 kg:uc008ajk.1 Cd68 657 Itch Eef1g 658 Elf1 Fbn1 659 Meis2 Hint1 660 Arid1a March5 661 Serping1 Usp48 662 Slc27a3 Hnf1b 663 Thoc2 Gga3 664 Gsta3 Drosha 665 Hnrnph2 Ubp1 666 Socs3 Pkn3 667 Armcx3 Tmem192 668 Siah1a Prpf31 669 kg:uc009ize.1 Hspd1 670 Irs2 Otub1 671 Mettl7a1 Mrpl20 672 Ppfibp2 Tead2 673 Blvrb Phpt1 674 Yipf5 Neu1 675 Plat Pygo2 676 Gm6578 Myeov2 677 Mat2b Cdk5 678 Tmpo Ndor1 679 Metap2 Rbp4 680 Zfp277 Psat1 681 Wls Mrpl41 682 Mesdc1 Snrpg 683 kg:uc009acs.1 Acot7 684 Col1a2 Vars 685 Csf1 Nono 686 Sulf2 Gtf2i 687 Ifrd1 Traf3 688 Wrnip1 Ppp2r4 689 Flii Actg2 690 2810474O19Rik Pi4k2a 691 Sep15 Slc35b2 692 2310030G06Rik Ubqln1 693 Cmtm3 Ppox 694 Mylip Bud31 695 Slc8a1 Man2b1 696 Btbd7 Nat15 697 Hdac5 Spon1 698 Zfand6 Cyc1 699 Tapbp Mpeg1 700 Keap1 Nsun2 701 Ube2n Rab4a 702 Ssr3 Mtmr11 703 H3f3a BC004004 704 Myst4 B4galnt1 705 G3bp1 Atp5k 706 Ugdh Lin37 707 Lamp2 D330041H03Rik 708 Zrsr1 Tbc1d17 709 Pim1 March6 710 Gm9199 2410015M20Rik 711 Supt16h 1810013D10Rik 712 Ano6 Eif2s1 713 Soat1 Traf7 714 Eci1 Rpl36al 715 Plce1 Psenen 716 Atg3 Aip 717 Bnc1 Cmas 718 Pik3c2a Rpia 719 Pqlc3 Ncbp1 720 Thrap3 Mea1 721 Irak4 Timm50 722 Kdm6b Ear12 723 Apol9a Fkbp1a 724 Wnt4 Commd4 725 1500003O03Rik Col5a3 726 Phf3 Fblim1 727 1110004F10Rik Cwh43 728 Kansl1 Arl2bp 729 Fth1 Mrpl46 730 Tmem50a Tcn2 731 Utp20 Add2 732 Smad4 Specc1l 733 Stmn2 Ppcs 734 Gstm1 Vrk3 735 Senp6 Trim25 736 Gda Nfatc1 737 Nucks1 Rap1gap 738 Ints10 Hsd17b12 739 Syne1 Epas1 740 Itga6 Ddx1 741 Acad9 Prdx6 742 Maged1 Mmp24 743 Spen Ndufb9 744 Chd1 Phf23 745 Taf3 Rpa2 746 Ptgs1 5031439G07Rik 747 Spare Rrp7a 748 R74862 Arfip2 749 B230120H23Rik Efna1 750 Tmem234 Agps 751 Ryk Sephs1 752 Dlgap4 Apoc2 753 Atp1b1 Mrps27 754 Parp14 Snn 755 Tgfbr2 Serinc3 756 Ccdc90a Pdcd5 757 Ncoa1 AA986860 758 Pppde1 Pitpna 759 Luc7l3 Vac14 760 Prg4 2810025M15Rik 761 Rab11fip1 Def8 762 Plk2 Hilpda 763 Ifi35 Eif6 764 Pdap1 Brd7 765 Cd248 Fes 766 Sesn1 Sbf1 767 Ecd Ak2 768 Ap1s3 1810035L17Rik 769 H2-K1 Lime1 770 Spag9 Hspe1 771 Tshz1 Csrp2bp 772 Dennd5a Uba5 773 Stag1 Gsta4 774 Gpx8 2900092E17Rik 775 Sod3 776 BC005561 777 kg:uc009vev.1 778 Ywhaz 779 Ganab 780 Rras2 781 Dusp14 782 kg:uc012hdk.1 783 Nr1d1 784 Wwc2 785 Ubxn2a 786 Iqsec1 787 kg:uc007vsr.1 788 Cfl1 789 Csrp1 790 Smchd1 791 Myl12a 792 Ubqln2 793 Tmcc3 794 Kdm5a 795 Rbm25 796 Wdr26 797 Vim 798 Arpc2 799 Calm1 800 Dnaja2 801 Shc1 802 Vps13a 803 Klf7 804 1810074P20Rik 805 BC003331 806 Itpr2 807 Jmjd1c 808 Pcdhgb5 809 Tubb2a 810 Ehd2 811 Ift74 812 Per1 813 Pitpnm2 814 Gstm4 815 Dnmt1 816 Tmco1 817 Lass4 818 Ptprf 819 Sirt2 820 Gfm2 821 Taf7 822 Spop 823 Zzef1 824 Ccdc34 825 Zfp281 826 Tuba1a 827 Ccdc109b 828 Cdk13 829 Dhx15 830 Src 831 Braf 832 Mapre2 833 Anxa7 834 Sept9 835 Alox12 836 Pknox1 837 2610034B18Rik 838 Topors 839 Phf21a 840 Qser1 841 Tirap 842 Fas 843 Lass2 844 6330406I15Rik 845 Parvb 846 Atp1a1 847 Mtmr6 848 Cd109 849 Dnajc1 850 Hp1bp3 851 1600029D21Rik 852 Ttc38 853 Mfhas1 854 Filip1l 855 Zfp148 856 Nkd1 857 Usp16 858 Tlr2 859 Zc3h18 860 Stk10 861 Ltbp4 862 Hdac3 863 Efhd2 864 Prkar2a 865 Atp6v1a 866 Sf3b4 867 Gprc5b 868 Clip3 869 Mettl2 870 Secisbp2 871 Fmod 872 kg:uc0091xf.1 873 Elovl6 874 Bzw1 875 Etfa 876 Hspa2 877 kg:uc007won.1 878 Rnf20

TABLE 3 Most Significant Gene Ontology Terms in CTC-c enriched genes using BP_FAT and CC_FAT Datasets q-value < 0.01 Odds Benjamini Source Term Count Ratio (q-value) GOTERM_BP_FAT GO: 0060429~epithelium development 35 2.92 8.72E−05 GOTERM_BP_FAT GO: 0030029~actin filament-based 27 3.47 6.85E−05 process GOTERM_BP_FAT GO: 0030036~actin cytoskeleton 26 3.57 4.95E−05 organization GOTERM_BP_FAT GO: 0007010~cytoskeleton organization 36 2.50 6.27E−04 GOTERM_BP_FAT GO: 0051173~positive regulation of 49 2.11 6.62E−04 nitrogen compound metabolic process GOTERM_BP_FAT GO: 0035295~tube development 31 2.66 7.80E−04 GOTERM_BP_FAT GO: 0010604~positive regulation of 54 1.93 0.001727 macromolecule metabolic process GOTERM_BP_FAT GO: 0031328~positive regulation of 49 2.01 0.0015751 cellular biosynthetic process GOTERM_BP_FAT GO: 0051789~response to protein stimulus 16 4.16 0.0014484 GOTERM_BP_FAT GO: 0035239~tube morphogenesis 23 3.05 0.0015064 GOTERM_BP_FAT GO: 0045449~regulation of transcription 140 1.42 0.0014097 GOTERM_BP_FAT GO: 0048729~tissue morphogenesis 28 2.66 0.0013058 GOTERM_BP_FAT GO: 0009891~positive regulation of 49 1.99 0.0012408 biosynthetic process GOTERM_BP_FAT GO: 0045935~positive regulation of 46 2.04 0.0012061 nucleobase, nucleoside, nucleotide and nucleic acid metabolic process GOTERM_BP_FAT GO: 0002009~morphogenesis of an 23 3.01 0.0012149 epithelium GOTERM_BP_FAT GO: 0048584~positive regulation of 24 2.92 0.0011396 response to stimulus GOTERM_BP_FAT GO: 0051276~chromosome organization 39 2.19 0.0012619 GOTERM_BP_FAT GO: 0045637~regulation of myeloid cell 12 5.33 0.0014358 differentiation GOTERM_BP_FAT GO: 0045785~positive regulation of cell 11 5.79 0.0016889 adhesion GOTERM_BP_FAT GO: 0045941~positive regulation of 43 2.05 0.0016795 transcription GOTERM_BP_FAT GO: 0045893~positive regulation of 39 2.12 0.0019852 transcription, DNA-dependent GOTERM_BP_FAT GO: 0051254~positive regulation of RNA 39 2.11 0.0022107 metabolic process GOTERM_BP_FAT GO: 0006357~regulation of transcription 51 1.87 0.0022801 from RNA polymerase II promoter GOTERM_BP_FAT GO: 0006325~chromatin organization 32 2.30 0.0025187 GOTERM_BP_FAT GO: 0010628~positive regulation of gene 43 2.00 0.0025252 expression GOTERM_BP_FAT GO: 0060562~epithelial tube 17 3.47 0.0025847 morphogenesis GOTERM_BP_FAT GO: 0042127~regulation of cell 45 1.89 0.0051485 proliferation GOTERM_BP_FAT GO: 0010557~positive regulation of 44 1.88 0.0071937 macromolecule biosynthetic process GOTERM_BP_FAT GO: 0002253~activation of immune 14 3.69 0.0078441 response GOTERM_BP_FAT GO: 0050778~positive regulation of 18 3.00 0.0080458 immune response GOTERM_BP_FAT GO: 0002684~positive regulation of 23 2.53 0.0088166 immune system process GOTERM_BP_FAT GO: 0045944~positive regulation of 33 2.09 0.0090124 transcription from RNA polymerase II promoter GOTERM_CC_FAT GO: 0005578~proteinaceous extracellular 32 2.38 0.0047511 matrix GOTERM_CC_FAT GO: 0031012~extracellular matrix 32 2.28 0.0051923 GOTERM_CC_FAT GO: 0044421~extracellular region part 60 1.71 0.0064365 GOTERM_CC_FAT GO: 0031981~nuclear lumen 65 1.62 0.0102413 GOTERM_CC_FAT GO: 0043233~organelle lumen 79 1.53 0.0085938 GOTERM_CC_FAT GO: 0005829~cytosol 45 1.81 0.0100772 GOTERM_CC_FAT GO: 0070013~intracellular organelle 78 1.52 0.0093866 lumen GOTERM_CC_FAT GO: 0031982~vesicle 43 1.83 0.0087123 GOTERM_CC_FAT GO: 0031974~membrane-enclosed lumen 80 1.50 0.0082696

TABLE 4 Most Significant Gene Sets Enriched in CTC-plt vs CTC-c q-value < 0.01 Odds Benjamini Source Term Count Ratio (q-value) GOTERM_BP_FAT GO: 0042060~wound healing 18 7.8 1.86E−07 GOTERM_BP_FAT GO: 0007596~blood coagulation 15 10.4 9.31E−08 GOTERM_BP_FAT GO: 0050817~coagulation 15 10.4 9.31E−08 GOTERM_BP_FAT GO: 0007599~hemostasis 15 10.3 7.59E−08 GOTERM_BP_FAT GO: 0050878~regulation of body fluid levels 15 8.2 1.30E−06 GOTERM_BP_FAT GO: 0030029~actin filament-based process 20 5.5 1.14E−06 GOTERM_BP_FAT GO: 0007010~cytoskeleton organization 26 3.9 3.95E−06 GOTERM_BP_FAT GO: 0030036~actin cytoskeleton organization 18 5.3 1.11E−05 GOTERM_BP_FAT GO: 0009611~response to wounding 26 3.6 1.02E−05 GOTERM_BP_FAT GO: 0007155~cell adhesion 33 2.9 2.86E−05 GOTERM_BP_FAT GO: 0022610~biological adhesion 33 2.8 2.70E−05 GOTERM_BP_FAT GO: 0001775~cell activation 19 3.7 4.70E−04 GOTERM_BP_FAT GO: 0030168~platelet activation 6 18.2 1.68E−03 GOTERM_BP_FAT GO: 0007229~integrin-mediated signaling 10 6.4 2.95E−03 pathway GOTERM_BP_FAT GO: 0016192~vesicle-mediated transport 25 2.6 3.81E−03 MSigDBv3.1 CGP GNATENKO PLATELET SIGNATURE 20 55.1 3.91E−24 MSigDBv3.1 CGP TENEDINI MEGAKARYOCYTE 14 15.3 1.35E−11 MARKERS MSigDBv3.1 REACTOME FACTORS INVOLVED IN 6 2.9 2.25E−02 CP:REACTOME MEGAKARYOCYTE DEVELOPMENT AND PLATELET PRODUCTION

TABLE 5 Most Significant Gene Sets Enriched in CTC-pro vs CTC-c q-value < 0.01 Odds Benjamini Source Term Count Ratio (q-value) GOTERM_BP_FAT GO: 0002495~antigen processing and 5 59.81 6.97E−04 presentation of peptide antigen via MHC class II GOTERM_BP_FAT GO: 0019886~antigen processing and 5 59.81 6.97E−04 presentation of exogenous peptide antigen via MHC class II GOTERM_BP_FAT GO: 0002504~antigen processing and 5 50.36 7.34E−04 presentation of peptide or polysaccharide antigen via MHC class II GOTERM_BP_FAT GO: 0002478~antigen processing and 5 41.60 1.10E−03 presentation of exogenous peptide antigen GOTERM_BP_FAT GO: 0019884~antigen processing and 5 34.18 1.87E−03 presentation of exogenous antigen GOTERM_BP_FAT GO: 0048002~antigen processing and 5 27.34 3.72E−03 presentation of peptide antigen GOTERM_BP_FAT GO: 0001775~cell activation 9 7.00 3.82E−03 GOTERM_BP_FAT GO: 0019882~antigen processing and 6 13.20 7.40E−03 presentation

Example 2 Supplemental Methods

Mice and cell lines. Mice with pancreatic cancer used in these experiments express Cre driven by Pdx1, LSL-Kras^(G12D), and Trp53^(lox/+) or Trp53^(lox/lox) (otherwise referred to as KPC) as previously described (Bardeesy et al., 2006). EGFP pancreatic lineage tagged KPC mice were generated by breeding the mT/mG mouse (Purchased from the Jackson Laboratory—Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J) into the breeder pairs used for KPC mouse generation. Normal FVB mice were purchased from Jackson Laboratory. All mice care and procedures were done under MGH SRAC approved protocols.

For cardiocentesis, animals were sedated with isofluorane, the chest wall was sterilized with ethanol and a skin incision was made above the rib cage to expose the thoracic cavity and eliminate normal skin epithelial cell contamination. A 23-gauge needle was used to draw approximately 1 mL of blood into a 1 mL syringe primed with 100 μL of PBS-10 mM EDTA pH 7.4 (Gibco). Blood EDTA concentration was raised to 5 mM by either the addition of a concentrated bolus of 500 mM EDTA or 1:1 dilution with 10 mM EDTA. Animals were then euthanized per animal protocol guidelines.

A mouse pancreatic cell line NB508 (Pdx1-Cre/Kras^(G12D)/Trp53^(lox/+)) previously generated from primary tumors developed in this endogenous model was GFP transfected by lentivirus (NB508-GFP). This cell line was used for spiked cell experiments and orthotopic tumor formation.

NB508-GFP Cell lines were maintained in standard culture conditions using RPMI-1640 medium+10% FBS+1% Pen/Strep (Gibco/Invitrogen).

For orthotopic experiments, NB508-GFP cells were orthotopically injected into the pancreas of healthy syngeneic (FVB background) mice. Briefly, mice were anesthesized with isofluorane and the left abdominal wall was treated with Nair® hair removal product, and sterilized with 70% ethanol. A small incision was made on the upper left lateral abdominal wall and the pancreas was mobilized. Approximately 1 million NB508-GFP cells in PBS in a total volume of 0.1 mL was injected into the pancreas. The peritoneum and abdominal wall was closed by sterile surgical staples. The tumors were allowed to grow for 2 weeks, at which time blood was obtained by cardiocentesis for CTC-iChip processing.

Adaptation of CTC Enrichment Technology.

Given the desire for an unbiased enrichment system, the negative depletion technology was selected for this application. All processing protocols were identical to those previously identified, except a rat anti-mouse CD45 antibody (BAM114, R&D Systems, USA) was conjugated to MyOne beads.

Spiked cell experiments were conducted to validate the system by spiking ˜1000 GFP expressing NB508 cells into 1 mL of healthy mouse blood and processing to determine recovery efficiency. Orthotopic models were used to validate recovery efficiency as well as initially determine expected depletion efficiency from tumor-bearing mice. In these experiments, enriched samples were evaluated for the number of GFP+ cells observed in the product.

Immunostaining of CTCs Isolated from the Endogenous Model.

Isolated CTCs were spun onto glass slides and immunostained using a primary-secondary approach. Primary antibodies were rabbit anti-wide spectrum cytokeratin (1:50, Abcam ab9377), and goat anti-mouse CD45 (1:500, R&D systems AF114). Secondary immunofluorescent-tagged antibodies were used for signal amplification. These were donkey anti-rabbit Alexa Fluor 594 (1:500, Invitrogen A-21207), and donkey anti-goat Alexa Fluor 488 (1:500, Invitrogen A-11055). Nuclei were then counterstained with DAPI and the slides were rinsed with PBS, cover slipped and stored at 4° C. They were imaged under 10× magnification using the BioView™ Ltd. automated imaging system (Billerica, Mass.) as well as an automated upright fluorescence microscope (Eclipse 90i™, Nikon, Melville, N.Y.). Positive staining for CK, without CD45 staining, was required for scoring potential CTCs, which were then manually reviewed. Threshold and baseline signals were established using specimens from non-tumor bearing mice.

Single Cell Micromanipulation.

After whole blood anti-CD45 negative depletion, the product containing enriched cells was collected in a 35 mm petri dish and viewed using a Nikon Eclipse Ti™ inverted fluorescent microscope. Cells of interest were identified based on intact cellular morphology and lack of labeling with anti-CD45 magnetic beads. These target cells were individually micromanipulated with a 10 μm transfer tip on an Eppendorf TransferMan® NK 2 micromanipulator and ejected into PCR tubes containing RNA protective lysis buffer (10×PCR Buffer II, 25 mM MgCl2, 10% NP40, 0.1 M DTT, SUPERase-In, Rnase Inhibitor, 0.5 uM UP1 Primer, 10 mM dNTP and Nuclease-free water) and immediately flash frozen in liquid nitrogen.

Single Cell Amplification and Sequencing.

Single cell amplification and sequencing were done as previously described (Tang et al., 2010) with slight modifications underlined below. RNA samples from extracted single circulating tumor cells were thawed on ice and incubated at 70° C. for 90 seconds. To generate cDNA, samples were treated with reverse transcription master mix (0.05 uL RNase inhibitor, 0.07 uL T4 gene 32 protein, and 0.33 uL SuperScript™ III Reverse Transcriptase per 1× volume) and incubated on thermocycler at 50° C. for 30 minutes and 70° C. for 15 minutes. To remove free primer, 1.0 uL of EXOSAP mix was added to each sample, which was incubated at 37° C. for 30 minutes and inactivated at 80° C. for 25 minutes. Next, a 3′-poly-A tail was added to the cDNA in each sample by incubating in master mix (0.6 uL 10×PCR Buffer II, 0.36 uL 25 mM MgCl₂, 0.18 uL 100 mM dATP, 0.3 uL Terminal Transferase, 0.3 uL RNase H, and 4.26 uL H₂O per 1× volume) at 37° C. for 15 minutes and inactivated at 70° C. for 10 minutes. A second strand cDNA was synthesis by dividing each sample into 4 and incubating in master mix (2.2 uL 10× High Fidelity PCR Buffer, 1.76 uL 2.5 mM each dNTP, 0.066 uL UP2 Primer at 100 uM, 0.88 uL 50 mM MgSO₄, 0.44 uL Platinum Taq DNA Polymerase, and 13.654 uL H₂O per 1× volume) at 95° C. for 3 minutes, 50° C. for 2 minutes, and 72° C. for 10 minutes.

PCR amplification (95° C. for 3 minutes, 20 cycles of 95° C. for 30 seconds, 67° C. for 1 minute, and 72° C. for 6 minutes 6 seconds) was performed with master mix (4.1 uL 10× High Fidelity PCR Buffer, 1.64 uL 50 mM MgSO₄, 4.1 uL 2.5 mM each dNTP, 0.82 uL AUP1 Primer at 100 uM, 0.82 uL AUP2 Primer at 100 uM, 0.82 uL Platinum Taq DNA Polymerase, and 6.7 uL H₂O per 1× volume). The 4 reactions of each sample were pooled and purified using the QIAGEN PCR Purification Kit (Cat. No 28106) and eluted in 50 uL EB buffer. Samples were selected by testing for genes Gapdh, ActB, Ptprc (CD45), Krt8, Krt18, Krt19, and Pdx1 using qPCR. Each sample was again divided in 4 and a second round of PCR amplification (9 cycles of 98° C. for 3 minutes, 67° C. for 1 minute, and 72° C. for 6 minutes 6 seconds) was performed with master mix (9 uL 10× High Fidelity PCR Buffer, 3.6 uL 50 mM MgSO₄, 13.5 uL 2.5 mM each dNTP, 0.9 uL AUP1 Primer at 100 uM, 0.9 uL AUP2 Primer at 100 uM, 1.8 uL Platinum Taq DNA Polymerase, and 59.1 uL H₂O per 1× volume). Samples were pooled and purified using Agencourt AMPure XP beads and eluted in 40 uL 1× low TE buffer.

Sequencing Library Construction.

To shear the DNA using the Covaris S2™ System, 1× low TE buffer and 1.2 uL shear buffer were added to each sample. Conditions of the shearing program include: 6 cycles, 5° C. bath temperature, 15° C. bath temperature limit, 10% duty cycle, intensity of 5, 100 cycles/burst, and 60 seconds. Then, samples were end-polished at room temperature for 30 minutes with master mix (40 uL 5× Reaction Buffer, 8 uL 10 mM dNTP, 8 uL End Polish Enzyme1, 10 uL End Polish Enzyme2, and 14 uL H₂O per 1× volume). DNA fragments larger than 500 bp were removed with 0.5× volumes of Agencourt AMPure XP™ beads. Supernatant was transferred to separate tubes. To size-select 200-500 bp DNA products, 0.3× volumes of beads were added and samples were washed 2× with 70% EtOH. The products were eluted in 36 uL low TE buffer. A dA-tail was added to each size-selected DNA by treating with master mix (10 uL 5× Reaction Buffer, 1 uL 10 mM dATP, and 5 uL A-Tailing Enzyme I per 1× volume) and incubated at 68° C. for 30 minutes and cooled to room temperature. To label and distinguish each DNA sample for sequencing, barcode adaptors (5500 SOLiD 4464405) were ligated to DNA using the 5500 SOLiD Fragment Library Enzyme Module™ (4464413). Following barcoding, samples were purified twice using the Agencourt AMPure XP™ beads and eluted in 22 uL low TE buffer. Following a round of PCR Amplification (95° C. for 5 minutes, 12 cycles of 95° C. for 15 seconds, 62° C. for 15 seconds, and 70° C. for 1 minute, and 70° C. for 5 minutes), the libraries were purified with AMPure XP beads. Finally, to quantify the amount of ligated DNA, SOLiD Library TaqMan Quantitation Kit™ was used to perform qPCR. Completed barcoded libraries were then subjected to emulsion PCR with template beads preparation and sequenced on the ABI 5500XL™.

RNA In Situ Hybridization (RNA-ISH).

Paraffin-embedded tissue blocks were freshly cut and frozen at −80° C. Upon removal from the freezer, slides were baked for 1 hr at 60° C. and fixed in %10 formaldehyde for 1 hr at room temperature (RT). Paraffin was removed using Histo-Clear™ and RNA-ISH™ was performed according to the Affymetrix QuantiGene ViewRNA ISH Tissue-2 Plex Assay™. Tissue sections were permeabilized by pretreating in buffer solution for 10 min at 95° C. and digested with protease for 10 min, before being fixed at RT in 5% formaldehyde. Target probe sets were applied and hybridized to the tissue by incubating for 2 hr at 40° C. Type 1 probes were used at a dilution of 1:50 and included Aldh1a2 (VB1-14197), Dcn (VB1-14962), Klf4 (VB1-14988), Igfbp5 (VB1-14987), and Sparc (VB1-14196). Type 6 probes included EGFP (VF6-13336) at 1:50 and pooled Krt8 (VB6-11060) and Krt18 (VB6-11059) at 1:100 each. Signal was amplified through the sequential hybridization of PreAmplifier and Amplifer QT mixes to the target probe set. Target mRNA molecules were detected by applying Type 6 Label Probe with Fast Blue substrate and Type 1 Label Probe with Fast Red substrate. Tissue was counterstained with Gill's Hemotoxylin for 10 sec at RT. DAPI (Invitrogen, D3571; 3.0 μg/ml) staining was performed for 1 min. Fluorescence microscopy using a Nikon 90i was used to visualize target mRNAs. Type 1 probes were detected in the Cy3 channel and Type 6 probes in the Cy5 channel. Merged images were generated using NIS-Elements™ software.

Determination of Reads-Per-Million (rpm)

Color space reads were aligned using Tophat™ version 2.0.4 (Trapnell et al., 2009) and Bowtie1™ version 0.12.7 with the no-novel-juncs argument set with mouse genome version mm9 and transcriptome defined by the mm9 knownGene table from genome.ucsc.edu. Reads that did not align or aligned to multiple locations in the genome were discarded. The mm9 table knownToLocusLink from genome.ucsc.edu was used to map, if possible, each aligned read to the gene who's exons the read had aligned to. The reads count for each gene was the number of reads that were so mapped to that gene. This count was divided by the total number of reads that were mapped to any gene and multiplied by one million to form the reads-per-million (rpm) count. Rpm rather than rpkm was used because a 3′ bias was noted in the alignments.

Unsupervised Hierarchical Clustering and Principal Components Analysis.

The minimum of 1 and the smallest positive value of the rpm matrix was added to the rpm matrix to eliminate zeros. The result was then log 10 transformed, yielding what is termed the log 10(rpm) matrix. The rows (corresponding to genes) of the log 10(rpm) matrix with the top 2000 standard deviations were retained and the rest of the rows discarded. The result was then median polished. The result was clustered using agglomerative hierarchical clustering with average linkage with distance metric equal to 1 minus the Pearson correlation coefficient. The principal components of the log 10(rpm) matrix were computed and the coordinates of the samples with respect to the first three principal components were plotted.

Measures of Cellular Heterogeneity.

For a collection of clusters of samples, a statistic, M, was defined as the mean over the clusters of the mean over all the pairs of samples in the cluster of the atan h of the correlation coefficient between the two columns of the rpm matrix corresponding to the pair. The “mean intra-cluster correlation coefficient” was defined as tan h(M). The jackknife estimator was used with respect to the samples to estimate a standard deviation, s, of the statistic. The 95% CI was defined as tan h (M±sφ⁻¹(0.975)), where 4 is the cumulative distribution function of the standard normal distribution. To compute a p-value for the null hypothesis that the mean of the distribution of the M statistic for a cluster is the same as the mean of the distribution of the M statistic for a collection of clusters, we let p=2(1−φ(|M1−M2|/√(s² ₁+s² ₂))). Of note, bootstrap was performed on the same data as an alternative to jackknife and similar results obtained (data not shown).

Supervised Differential Gene Expression Using Rank Product.

To find differentially expressed genes between two sets of samples, analysis was begin with the log 10(rpm) matrix defined above. Columns corresponding to samples not in either set of samples were removed. Then removed rows for which the 90^(th) percentile of the values was less than log 10(10) were removed. The RP function of the Bioconductor (Gentleman et al., 2004) RankProd™ package (version 2.28.0) was used to get FDR estimates for both up and down differential expression. Genes were considered to be differentially expressed if their FDR estimate was less than 0.01, but discarded if they were both up and down differentially expressed, if there were any.

Gene Set Enrichment.

Enrichment was considered in four gene set collections: (1) all of KEGG™, as found in DAVID™ 6.7 (Huang da et al., 2009), (2) Gene Ontology (GO) using GO_BP as found in DAVID 6.7, and (3) GO_CC as found in DAVID 6.7. Sets of genes found to be differentially expressed were tested for enrichment in the gene set collections using a hypergeometric test for each gene set in the collection. The resulting p-values for each collection were converted to FDR estimates using the Benjamini-Hochberg method (Benjamini and Hochberg, 1995).

Digital Removal of all Annotated Platelet Transcripts

The 446 genes whose expression in the log 10(rpm) matrix had an absolute value of correlation coefficient greater than 0.6 with the expression of any of the genes in the gene sets named GNATENKO_PLATELET_SIGNATURE and TENEDINI_MEGAKARYOCYTE_MARKERS in MSigDB v3.1 were removed from the log 10(rpm) matrix (defined above). Clustering was then performed as described above.

SUPPLEMENTAL METHODS REFERENCES

-   Bardeesy, N., Aguirre, A. J., Chu, G. C., Cheng, K. H., Lopez, L.     V., Hezel, A. F., Feng, B., Brennan, C., Weissleder, R., Mahmood,     U., et al. (2006). Both p16(Ink4a) and the p19(Arf)-p53 pathway     constrain progression of pancreatic adenocarcinoma in the mouse.     Proc Natl Acad Sci USA 103, 5947-5952. -   Benjamini, Y., and Hochberg, Y. (1995). Controlling the False     Discovery Rate: A Practical and Powerful Approach to Multiple     Testing. Journal of the Royal Statistical Society Series B     (Methodological) 57, 289-300. -   Gentleman, R. C., Carey, V. J., Bates, D. M., Bolstad, B., Dettling,     M., Dudoit, S., Ellis, B., Gautier, L., Ge, Y., Gentry, J., et al.     (2004). Bioconductor: open software development for computational     biology and bioinformatics. Genome biology 5, R80. -   Huang da, W., Sherman, B. T., and Lempicki, R. A. (2009). Systematic     and integrative analysis of large gene lists using DAVID     bioinformatics resources. Nat Protoc 4, 44-57. -   Tang, F., Barbacioru, C., Nordman, E., Li, B., Xu, N., Bashkirov, V.     I., Lao, K., and Surani, M. A. (2010). RNA-Seq analysis to capture     the transcriptome landscape of a single cell. Nat Protoc 5, 516-535. -   Trapnell, C., Pachter, L., and Salzberg, S. L. (2009). TopHat:     discovering splice junctions with RNA-Seq. Bioinformatics 25,     1105-1111.

Example 3

A comparative analysis of mouse pancreatic CTCs indicated an enrichment of 60 extracellular proteins (Table 6). Evaluation of these particular biomarkers and therapeutic targets was undertaken in human pancreatic circulating tumor cells and the most abundant targets in human pancreatic CTCs are shown (FIG. 7). These not only represent potential biomarkers, but given their nature as proteins on the external surface of tumor cells, they are therapeutic targets. The extracellular proteins of Table 6 can be targeted, e.g. by antibody-based therapeutics (e.g. as in the cases of trastuzumab for HER2, cetuximab for EGFR, and bevacizumab for VEGF) to treat cancer.

TABLE 6 List of Pancreatic CTC enriched Extracellular Proteins. OFFICIAL GENE SYMBOL Gene Name Abi3bp ABI gene family, member 3 (NESH) binding protein Adamts5 similar to a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 5 (aggrecanase-2); a disintegrin-like and metallopeptidase (reprolysin type) with thrombospondin type 1 motif, 5 (aggrecanase-2) Adamtsl1 ADAMTS-like 1 Ang angiogenin, ribonuclease, RNase A family, 5 Arsa arylsulfatase A C1rl complement component 1, r subcomponent-like C3 complement component 3; similar to complement component C3 prepropeptide, last C4a similar to Complement C4 precursor; complement component 4A (Rodgers blood group); similar to complement C4; complement component 4B (Childo blood group) C4b similar to Complement C4 precursor; complement component 4A (Rodgers blood group); similar to complement C4; complement component 4B (Childo blood group) Ccdc80 coiled-coil domain containing 80 Cd109 CD109 antigen Chi3l1 chitinase 3-like 1 Clec3b C-type lectin domain family 3, member b Cmtm3 CKLF-like MARVEL transmembrane domain containing 3 Cmtm7 CKLF-like MARVEL transmembrane domain containing 7 Col14a1 collagen, type XIV, alpha 1 Col1a2 collagen, type I, alpha 2 Col3a1 collagen, type III, alpha 1 Col4a6 collagen, type IV, alpha 6 Csf1 colony stimulating factor 1 (macrophage) Dag1 dystroglycan 1 Dcn decorin Dmkn dermokine Fbln1 fibulin 1 Fgf1 fibroblast growth factor 1 Fmod fibromodulin Gpc3 glypican 3 Gpc4 glypican 4; similar to Glypican 4 Hmgb1 high mobility group box 1 Ifnar2 interferon (alpha and beta) receptor 2 Igfbp5 insulin-like growth factor binding protein 5 Il16 interleukin 16 Lama4 laminin, alpha 4 Ltbp4 latent transforming growth factor beta binding protein 4 Mfap1a similar to microfibrillar-associated protein 1A; microfibrillar-associated protein 1A; microfibrillar-associated protein 1B Nid2 nidogen 2 Ogn osteoglycin Pdap1 PDGFA associated protein 1 Pf4 platelet factor 4 Plat plasminogen activator, tissue Podn podocan Prelp proline arginine-rich end leucine-rich repeat Rspo1 R-spondin homolog (Xenopus laevis) Serping1 serine (or cysteine) peptidase inhibitor, clade G, member 1 Slurp1 secreted Ly6/Plaur domain containing 1 Sod3 superoxide dismutase 3, extracellular Sparc secreted acidic cysteine rich glycoprotein; similar to Secreted acidic cysteine rich glycoprotein Spock2 sparc/osteonectin, cwcv and kazal-like domains proteoglycan 2 Spon2 spondin 2, extracellular matrix protein Sulf1 sulfatase 1 Sulf2 sulfatase 2 Tgfb2 transforming growth factor, beta 2 Tgm2 transglutaminase 2, C polypeptide Thbd thrombomodulin Thbs1 thrombospondin 1; similar to thrombospondin 1 Thsd4 thrombospondin, type I, domain containing 4 Timp2 tissue inhibitor of metalloproteinase 2 Tnxb tenascin XB Tpt1 predicted gene 1974; tumor protein, translationally-controlled 1 pseudogene; tumor protein, translationally-controlled 1; predicted gene 14456 Twsg1 twisted gastrulation homolog 1 (Drosophila) Wnt4 wingless-related MMTV integration site 4

Extending these CTC enriched genes to human pancreatic, breast, and prostate single cell CTC data identified 5 candidate genes shown in Table 9.

TABLE 9 Percent of human single CTCs with high expression by RNA-seq Percent of Single CTCs >50 RPM of Expression Pancreas Breast Prostate ALL Cancer Type (N = 7) (N = 29) (N = 77) (N = 113) TPT1 86% 90% 90% 89% HMGB1 43% 62% 44% 49% SPON2 43% 7% 45% 35% SPARC 100% 41% 9% 23% ARSA 71% 17% 5% 12%

Focusing on pancreatic cancer, SPARC was selected as an initial gene to evaluate. SPARC RNA-ISH in mouse and human primary tumors (data not shown) demonstrated significant expression in the stromal cells of the tumor that provides essential microenvironmental signals to tumors. Much effort in the field focuses on targeting the stroma of PDAC for therapeutic efficacy [1-4] making SPARC a CTC therapeutic target as well as a stromal directed target. A total of 196/198 (99%) of human pancreatic tumors were positive for SPARC and 36% with clear epithelial tumor cell expression.

Evaluation of human pancreatic cancer cell lines identified 3 of 5 cell lines with elevated SPARC expression which correlates to increased migratory behavior, a surrogate in vitro assay that correlates with metastatic behavior (FIG. 8).

Evaluation of SPARC function in human pancreatic cancer was done using short hairpin RNA interferences (shRNA) on the two cell lines with highest SPARC expression (PDAC2 and PDAC3). Multiple in vitro assays were done including proliferation, migration, invasion, scratch, and soft agar. The most profound effects of suppressing SPARC expression was on migratory behavior (FIG. 9 and data not shown), indicating SPARC is not only present in many CTCs, but has functional consequences when inhibited in cell line models.

Given these data, in vivo tail vein inoculation was performed using PDAC-3 to determine if SPARC knockdown affected metastasis. Initial data at 2 weeks post tail vein injection indicates there is reduced metastatic potential when SPARC is inhibited by shRNA with 83% of control mice with metastases compared to 40% in cell lines with shRNA against SPARC (FIG. 10).

Surface Protein Targets

Most of the targets identified in Table 9 are secreted factors and analysis of genes annotated as cell surface proteins are summarized in Table 14.

TABLE 14 Percent of human single CTCs with high expression of surface protein genes Percent of Single CTCs >50 RPM of Expression Cancer Pancreas Breast Prostate type (N = 7) (N = 29) (N = 77) ALL (N = 113) IL6ST 0% 38% 8% 15% ARSA 71% 17% 5% 12% TIMP2 0% 21% 4% 8% CD55 0% 17% 4% 7% SULF2 0% 24% 0% 6% ITGA6 0% 14% 3% 5% SDC4 0% 14% 3% 5% CDON 0% 7% 5% 5% SV2A 14% 3% 1% 3%

It is contemplated herein that these genes are targets given they would be integrated into the plasma membrane of CTCs. In general, RNA expression of cell surface markers tend to be lower than actual protein levels on cells.

Contemplated herein are antibodies to IL6ST, SULF2, and SV2A for therapeutic utility.

1. IL6ST—signal transducer for IL6, LIF, CNTF, and oncostatin M.

-   -   a. Important for STAT3 activation downstream     -   b. Antibodies against IL6 receptor and IL6 have been developed         for human disease including cancer

2. SULF2—sulfatase modifies heparin sulfate by removing 6-O-sulfate groups

-   -   a. Expression enriched in cancer progression and metastasis     -   b. Drugs have been developed against sulfatase activity and         tested with activity in liver cancer models

3. SV2A—synaptic vesicle glycoprotein elevated in neuroendocrine cells

-   -   a. A marker of neuroendocrine cells, which appear at the         epithelial stromal border of human pancreatic cancer     -   b. Neuroendocrine differentiation common feature in cancers and         portends to more aggressive disease

REFERENCES

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Example 5

Circulating tumor cells (CTCs) are shed from primary tumors into the bloodstream, mediating the hematogenous spread of cancer to distant organs. To define their composition, genomewide expression profiles of CTCs were compared with matched primary tumors in a mouse model of pancreatic cancer, isolating individual CTCs using epitope-independent microfluidic capture, followed by single-cell RNA sequencing. CTCs clustered separately from primary tumors and tumor-derived cell lines, showing low proliferative signatures, enrichment for Aldh1a2, biphenotypic expression of epithelial and mesenchymal markers, and expression of Igfbp5, a gene transcript enriched at the epithelial-stromal interface. Mouse as well as human pancreatic CTCs exhibit a very high expression of stromal-derived extracellular matrix (ECM) proteins, including SPARC, whose knockdown in cancer cells suppresses cell migration and invasiveness. The aberrant expression by CTCs of stromal ECM genes points to their contribution of microenvironmental signals for the spread of cancer to distant organs.

Classical CTCs expressed predominantly the Aldh1a2 isoform, while Aldh1a1 was expressed in a variety of cell types (data not shown). Within single CTCs, there was no correlation between expression of Aldh1 isoforms and either enrichment for the mesenchymal genes (Cdh11, Vim) or loss of epithelial genes (Cdh1, Muc1), indicating that stem cell and EMT markers are not intrinsically linked in CTCs. Analysis of primary pancreatic tumors for Aldh1a2 using RNA in situ hybridization (RNA-ISH) identified rare epithelial tumor cells expressing this stem cell marker, but the majority of expression was present within the cancer associated stromal cells (FIG. 12A), consistent with immunohistochemistry for ALDH protein in human PDAC (Rasheed et al., 2010).

Besides the evident diversity of CTCs, shared transcripts were searched for that could provide further insight into their cell of origin within the primary tumor and the mechanisms by which they invade and survive within the bloodstream and ultimately identify potential CTC-specific therapeutic targets. Rigorous criteria were selected to identify the most highly enriched CTC-c transcripts (RP score<300), expressed at very high levels (>100 rpm) in R90% of all classical CTCs. Three genes met these criteria: Kruppel-like factor 4 (Klf4), one of the key stem cell (iPS) reprogramming factors (Takahashi and Yamanaka, 2006), insulin-like growth factor binding protein 5 (Igfbp5), an extracellular growth factor binding protein and decorin (Dcn). RNA-ISH was utilized in primary tumor specimens to identify the potential colocalization of these three highly enriched CTC genes. In contrast to Aldh1a2, Klf4 is expressed in epithelial components of the primary tumor (FIG. 12B). Igfbp5 is of particular interest, in that it is expressed focally at the tumor epithelial-stromal interface (FIG. 12C). It is contemplated herein that this geographic area is enriched for cancer cells undergoing EMT, contributing to the mixed epithelial/stromal transcriptional programs evident by RNA-seq of single CTCs.

In addition to highly expressing Dcn, CTCs consistently had high levels of multiple ECM gene transcripts. GO analysis of all CTC-enriched genes (Table 3) identified 32 proteinaceous ECM genes (GO:0005578, OR 2.4, q-value 4.8 3 10.3). These genes are normally expressed in reactive stromal cells, rather than in epithelial cancer cells, and while recent studies have highlighted the importance of the stroma in supporting pancreatic cancer pathogenesis and metastasis (Feig et al., 2012; Neesse et al., 2011, 2013; Olive et al., 2009; Provenzano et al., 2012), the expression of these stroma-associated ECM genes within tumor cells in circulation was unexpected. Using RP differential expression analysis, CTCs were compared with purified EGFP-tagged primary tumor single cells (TuGMP3) and bulk tumor samples (tumor cells admixed with reactive stromal cells). Six proteinaceous ECM genes were highly expressed by CTCs and by stromal component, but not by epithelial cells within primary tumors: Dcn, Sparc, Ccdc80, Col1a2, Col3a1, and Timp2 (data not shown). RNA-ISH analysis of both Dcn and Spare confirmed diffuse expression in stromal elements of mouse primary tumors, with rare areas where these transcripts are colocalized with keratin-expressing cells at the epithelial-stromal border (data not shown).

SPARC is a ECM protein gene. RNA-ISH analysis of 198 primary human PDACs demonstrates abundant stromal cell expression of SPARC transcripts in 99% of cases, with up to a third of tumors with rare epithelial cells expressing this ECM gene product (data not shown). Consistent with these observations, RNA-seq of EGFP-tagged single primary tumor cells (data not shown) identified only 1 of 20 cells (5%) with coexpression of high levels (>100 rpm) of Spare and Krt19.

In summary, abundant expression of ECM genes is a common feature of all keratin-rich classical CTCs. This is in marked contrast to the primary tumor, where these gene products are secreted by supporting stromal cells and not by the epithelial cancer cells. However, rare cells at the epithelial-stromal interface of primary tumors do appear to express both keratins and ECM genes, consistent with the pattern observed in CTCs themselves.

To confirm the expression of proteinaceous ECM genes by human cancer cells circulating in the bloodstream, single CTCs were isolated from patients with pancreatic (n=7), breast (n=29), and prostate (n=77) cancers and subjected these to single-cell RNA-seq. Six ECM protein genes were highly expressed in human CTCs (>100 rpm in >15% of all CTC samples) (FIG. 13; Table 13). Notably, three genes (SPARC, MGP, SPON2) are ECM glycoproteins, defined as part of the core matrisome (Naba et al., 2012). The core matrisome protein SPARC was particularly enriched in pancreatic CTCs being expressed at high levels (>100 rpm) in 100% of pancreatic CTCs compared to 31% of breast and 9% of prostate CTCs. The notable differences in ECM protein gene expression across human epithelial CTCs suggest microenvironment tissue specificity as well as probable redundancies in ECM protein signaling. Together, the consistent expression of ECM gene family members in human CTCs indicates that their upregulation contributes either to the generation of CTCs from primary tumors or to the survival of cancer cells deprived of microenvironmental signals as they circulate in the bloodstream.

In order to define the functional consequences of SPARC expression in pancreatic cancer cells, a panel of patient-derived, low-passage PDAC cell lines was screened for expression. Two human PDAC cell lines with relatively high SPARC expression were identified (PDAC2 and PDAC3), making it possible to test the consequences of small hairpin RNA (shRNA)-mediated knockdown (FIG. 8, 9, FIGS. 16A-16D). Suppression of endogenous SPARC expression in both PDAC2 and PDAC3 cell lines using two independent shRNA constructs did not affect proliferation in 2D cultures or anchorage-independent tumor sphere formation (FIGS. 14A-14B, FIGS. 16A-16D). However, SPARC knockdown by both shRNAs significantly reduced pancreatic cancer cell migration in wound scratch assays and their invasive properties, as measured by in vitro Boyden assays (data not shown).

Tail vein injection of SPARC-suppressed PDAC3 cells using both shRNA constructs generated significantly fewer lung metastases than cells expressing nontargeting hairpin (shNT) controls (FIG. 14D). Metastases generated from orthotopic pancreatic xenografts were also significantly reduced for SPARC-suppressed PDAC3 cells, as measured by luciferase imaging and normalized for primary tumor size (FIG. 14E). Thus, SPARC expression by pancreatic cancer cells appears to selectively enhance their invasive and migratory properties to augment metastatic virulence. The high levels of SPARC expression evident in virtually all pancreatic CTCs thus raises the possibility that it contributes significantly to the metastatic spread of pancreatic cancer.

Discussion

Described herein is the detailed analysis of CTC composition and diversity in pancreatic cancer, using single-cell RNA-seq. High-quality transcriptomes were achieved in 93 single mouse pancreatic CTCs, which were compared with bulk and single-cell preparations from matched primary tumors and from an immortalized cell line established from the same mouse pancreatic tumor model. The use of the KPC mouse model made it possible to compare simultaneously isolated primary tumor specimens and CTCs, and it allowed measurements of CTC heterogeneityacross multiple mice sharing the same Kras/Trp53 genetic drivers. The large number of isolated CTCs and the high quality of the isolated RNA from these cells reflect the application of the CTC-iChip technology, which effectively depletes normal blood components, enriching for CTCs that are untagged and accessible for single-cell manipulation. Finally, the purification of CTCs irrespective of their cell-surface epitopes avoids any bias associated with their purification based on expression of common epithelial markers such as EpCAM.

Together, the observations made herein include the following. (1) CTC expression profiles cluster into three classes, including a major “classical CTC” group, and others that are defined by platelet derived markers or proliferative signatures. (2) Common features shared by virtually all classical CTCs include expression of both epithelial and mesenchymal markers, the stem cell-associated gene Aldh1a2, and three highly expressed transcripts, Klf4, Igfbp5, and Dcn. The specific localization of Igfbp5-expressing cells at the epithelial-stromal boundary within primary tumors may point to a region that contributes significantly to CTC generation. (3) The most highly enriched CTC-specific transcripts shared by almost all classical CTCs encode extracellular matrix proteins, such as Sparc. (4) Aberrant expression in CTCs of this ECM gene product, which is normally abundant in the tumor stromal compartment, is observed in both mouse and human pancreatic CTCs, and its knockdown attenuates cancer cell migration and invasion in reconstituted systems. (FIG. 15) Compared with RNA-seq of partially purified, bulk CTC populations, which required digital subtraction of leukocyte-derived reads (Yu et al., 2012, 2013), the single-cell analysis reported here provides considerably more depth of tumor cell-specific transcript reads, and it allows measurements of CTC heterogeneity.

It is contemplated herein that in addition to the initiating mutations, somatically acquired genetic and epigenetic changes may distinguish CTCs derived from different tumors. Multiple mouse tumors contributed to each of the three distinct clusters of CTCs. Despite their atypical expression pattern, the identification of platelet-associated and proliferative CTC subsets as being tumor-derived is established by their inclusion of lineage-tagged tumor cells. The more characteristic expression pattern exhibited by the classical CTC cluster enabled detailed comparison with primary tumor cells, thereby providing further insight into the origin and properties of CTCs.

Mouse pancreatic classical CTCs uniformly lose expression of the epithelial marker E-cadherin (Cdh1), a key feature of epithelial-to-mesenchymal transition. However, the cells do not lose expression of other epithelial markers, such as cytokeratins, nor is there a consistent increase in classical mesenchymal markers such as vimentin. As such, most classical CTCs appear arrested in a biphenotypic state. Despite their expression of cytokeratins, which are present in the epithelial components of the primary tumor, most other highly expressed markers in CTCs are shared with the stromal component of the primary tumor. Among these stromal genes is Aldh1a2 (Rasheed and Matsui, 2012; Rasheed et al., 2010). A provocative observation relating to the shared epithelial and mesenchymal state of classical CTCs is their virtually universal (93%) expression of Igfbp5, which is uniquely expressed in a small subpopulation of cells at the epithelial/stromal interface within primary tumors. This raises the possibility that this critical location within the primary tumor generates a disproportionate fraction of viable CTCs.

The most unexpected observation from the single-CTC RNAseq study is the high abundance of ECM transcripts in the vast majority of classical CTCs. The coexpression of pancreatic cancer-enriched cytokeratins (Krt7 and Krt19) in single cells expressing these ECM gene products excludes the possibility that these represent circulating tumor-derived fibroblasts.

Consistent with the aberrant expression of SPARC in some pancreatic cancer cells, a subset of patient-derived tumor cell lines also coexpress it along with epithelial cytokeratins. The reduction in cell migration and metastatic potential exhibited by these pancreatic cell lines following SPARC knockdown indicates that it contributes to CTC-mediated metastasis. It is contemplated herein that Sparc expression contributes to metastasis, but inherent redundancies in ECM protein expression may mitigate this effect in some embodiments.

Considerable effort has been directed to targeting the pancreatic cancer stroma as a means of improving delivery of chemotherapeutics as well as stripping tumor cells of their supportive microenvironment (Neesse et al., 2011; Olive et al., 2009; Provenzano et al., 2012; Rasheed et al., 2012). The findings described herein, e.g., that these gene products are also expressed by CTCs themselves suggests a remarkable level of cellular plasticity. To the extent that invasive properties of CTCs are mediated in part by expression of such ECM proteins, it also raises the possibility of targeting cancer cells in the blood.

TABLE 13 Human CTC ECM Gene Expression Percent of Samples >100 RPM ECM Gene Prostate Count Symbol All CTCs PDAC CTCs Breast CTCs CTCs 1 ANXA2 36.3% 0.0% 51.7% 33.8% 2 SPON2 29.2% 0.0% 3.4% 41.6% 3 LGALS3 22.1% 42.9% 37.9% 14.3% 4 SPARC 21.2% 100.0% 31.0% 10.4% 5 LGALS3BP 16.8% 0.0% 34.5% 11.7% 6 MGP 15.9% 57.1% 44.8% 1.3% 7 LAMC1 15.0% 0.0% 6.9% 19.5% 8 SMC3 15.0% 42.9% 17.2% 11.7% 9 CALR 14.2% 0.0% 6.9% 18.2% 10 TIMP1 13.3% 14.3% 27.6% 7.8% 11 MMP24 11.5% 0.0% 10.3% 13.0% 12 DAG1 10.6% 0.0% 20.7% 7.8% 13 ERBB2IP 10.6% 14.3% 20.7% 6.5% 14 MMP19 10.6% 0.0% 10.3% 11.7% 15 AGRN 8.8% 0.0% 6.9% 10.4% 16 CRTAP 8.8% 0.0% 6.9% 10.4% 17 COL24A1 8.0% 57.1% 17.2% 0.0% 18 ANG 7.1% 0.0% 0.0% 10.4% 19 MFAP1 7.1% 0.0% 6.9% 7.8% 20 VWF 7.1% 14.3% 17.2% 2.6% 21 VWA1 7.1% 0.0% 3.4% 9.1% 22 TIMP2 6.2% 0.0% 13.8% 3.9% 23 ECM1 6.2% 0.0% 24.1% 0.0% 24 LTBP1 6.2% 28.6% 10.3% 2.6% 25 LGALS1 6.2% 0.0% 10.3% 5.2% 26 SERPINA1 6.2% 0.0% 20.7% 1.3% 27 SPOCK1 6.2% 14.3% 0.0% 7.8% 28 TFF3 6.2% 0.0% 17.2% 2.6% 29 NPNT 5.3% 0.0% 3.4% 6.5% 30 TFIP11 5.3% 14.3% 6.9% 3.9% 31 COL9A2 4.4% 0.0% 0.0% 6.5% 32 COL6A1 4.4% 0.0% 0.0% 6.5% 33 FN1 4.4% 14.3% 10.3% 1.3% 34 LAD1 4.4% 0.0% 10.3% 2.6% 35 LAMA1 4.4% 14.3% 3.4% 3.9% 36 LAMB2 4.4% 0.0% 10.3% 2.6% 37 MATN2 4.4% 14.3% 3.4% 3.9% 38 ZP3 4.4% 0.0% 0.0% 6.5% 39 ADAMTSL3 3.5% 28.6% 3.4% 1.3% 40 FRAS1 3.5% 14.3% 0.0% 3.9% 41 TIMP3 3.5% 0.0% 3.4% 3.9% 42 DST 3.5% 0.0% 6.9% 2.6% 43 GFOD2 3.5% 14.3% 0.0% 3.9% 44 LAMA3 3.5% 14.3% 0.0% 3.9% 45 LAMB1 3.5% 14.3% 0.0% 3.9% 46 MMP7 3.5% 0.0% 0.0% 5.2% 47 ANGPTL4 2.7% 0.0% 0.0% 3.9% 48 BMP4 2.7% 0.0% 0.0% 3.9% 49 LTBP2 2.7% 28.6% 3.4% 0.0% 50 LEPRE1 2.7% 0.0% 0.0% 3.9% 51 LUM 2.7% 0.0% 0.0% 3.9% 52 NID2 2.7% 14.3% 6.9% 0.0% 53 SLC1A3 2.7% 28.6% 0.0% 1.3% 54 TECTA 2.7% 14.3% 3.4% 1.3% 55 THSD4 2.7% 0.0% 6.9% 1.3% 56 ADAMTS15 1.8% 0.0% 6.9% 0.0% 57 USH2A 1.8% 14.3% 3.4% 0.0% 58 APLP1 1.8% 0.0% 0.0% 2.6% 59 COL4A3 1.8% 14.3% 3.4% 0.0% 60 COL7A1 1.8% 0.0% 3.4% 1.3% 61 COL11A1 1.8% 0.0% 6.9% 0.0% 62 COL11A2 1.8% 0.0% 0.0% 2.6% 63 COL15A1 1.8% 28.6% 0.0% 0.0% 64 CTGF 1.8% 0.0% 0.0% 2.6% 65 CRISP3 1.8% 0.0% 0.0% 2.6% 66 DCN 1.8% 0.0% 0.0% 2.6% 67 ENTPD2 1.8% 0.0% 0.0% 2.6% 68 FMOD 1.8% 0.0% 3.4% 1.3% 69 GPC1 1.8% 0.0% 0.0% 2.6% 70 HSPG2 1.8% 0.0% 0.0% 2.6% 71 LAMA5 1.8% 0.0% 3.4% 1.3% 72 LAMC2 1.8% 14.3% 0.0% 1.3% 73 MMP10 1.8% 0.0% 3.4% 1.3% 74 MMP12 1.8% 0.0% 0.0% 2.6% 75 NTN4 1.8% 0.0% 6.9% 0.0% 76 NAV2 1.8% 0.0% 6.9% 0.0% 77 PAPLN 1.8% 0.0% 3.4% 1.3% 78 SFTPA2 1.8% 0.0% 0.0% 2.6% 79 VCAN 1.8% 14.3% 0.0% 1.3% 80 ADAMTS13 0.9% 0.0% 3.4% 0.0% 81 ADAMTS3 0.9% 14.3% 0.0% 0.0% 82 ADAMTS5 0.9% 14.3% 0.0% 0.0% 83 ADAMTSL4 0.9% 0.0% 0.0% 1.3% 84 EFEMP1 0.9% 0.0% 3.4% 0.0% 85 EFEMP2 0.9% 0.0% 3.4% 0.0% 86 EGFLAM 0.9% 14.3% 0.0% 0.0% 87 KAL1 0.9% 0.0% 0.0% 1.3% 88 KAZALD1 0.9% 0.0% 0.0% 1.3% 89 MAMDC2 0.9% 14.3% 0.0% 0.0% 90 SMOC1 0.9% 0.0% 0.0% 1.3% 91 SMOC2 0.9% 0.0% 0.0% 1.3% 92 ACHE 0.9% 0.0% 0.0% 1.3% 93 AMTN 0.9% 0.0% 3.4% 0.0% 94 ANXA2P2 0.9% 0.0% 3.4% 0.0% 95 CPZ 0.9% 0.0% 3.4% 0.0% 96 CHADL 0.9% 0.0% 0.0% 1.3% 97 COCH 0.9% 0.0% 0.0% 1.3% 98 COL6A6 0.9% 14.3% 0.0% 0.0% 99 COL1A2 0.9% 0.0% 3.4% 0.0% 100 COL2A1 0.9% 0.0% 0.0% 1.3% 101 COL4A1 0.9% 14.3% 0.0% 0.0% 102 COL4A2 0.9% 0.0% 0.0% 1.3% 103 COL4A6 0.9% 0.0% 0.0% 1.3% 104 COL5A1 0.9% 14.3% 0.0% 0.0% 105 COL6A2 0.9% 0.0% 0.0% 1.3% 106 COL8A1 0.9% 14.3% 0.0% 0.0% 107 COL12A1 0.9% 14.3% 0.0% 0.0% 108 COL14A1 0.9% 14.3% 0.0% 0.0% 109 COL19A1 0.9% 14.3% 0.0% 0.0% 110 COL17A1 0.9% 14.3% 0.0% 0.0% 111 COL22A1 0.9% 14.3% 0.0% 0.0% 112 ENTPD1 0.9% 14.3% 0.0% 0.0% 113 FBN2 0.9% 0.0% 0.0% 1.3% 114 FBN3 0.9% 0.0% 3.4% 0.0% 115 FBLN1 0.9% 14.3% 0.0% 0.0% 116 FBLN7 0.9% 0.0% 0.0% 1.3% 117 GPC4 0.9% 0.0% 3.4% 0.0% 118 HMCN1 0.9% 14.3% 0.0% 0.0% 119 IMPG1 0.9% 14.3% 0.0% 0.0% 120 IMPG2 0.9% 0.0% 3.4% 0.0% 121 LAMA2 0.9% 0.0% 3.4% 0.0% 122 LAMB3 0.9% 14.3% 0.0% 0.0% 123 MEPE 0.9% 0.0% 3.4% 0.0% 124 MMP1 0.9% 14.3% 0.0% 0.0% 125 MMP2 0.9% 0.0% 3.4% 0.0% 126 MMP25 0.9% 0.0% 0.0% 1.3% 127 MMP3 0.9% 0.0% 3.4% 0.0% 128 MMP9 0.9% 14.3% 0.0% 0.0% 129 OGN 0.9% 14.3% 0.0% 0.0% 130 PI3 0.9% 0.0% 0.0% 1.3% 131 PRELP 0.9% 14.3% 0.0% 0.0% 132 PTPRZ1 0.9% 14.3% 0.0% 0.0% 133 RELN 0.9% 0.0% 3.4% 0.0% 134 ADAMTSL2 0.9% 0.0% 0.0% 1.3% 135 TGFBI 0.9% 0.0% 3.4% 0.0% 136 UCMA 0.9% 0.0% 3.4% 0.0% 137 VIT 0.9% 0.0% 3.4% 0.0% 138 WNT10A 0.9% 14.3% 0.0% 0.0% 139 WNT10B 0.9% 0.0% 0.0% 1.3% 140 WNT11 0.9% 0.0% 3.4% 0.0% 141 WNT4 0.9% 0.0% 0.0% 1.3% 142 ZP2 0.9% 14.3% 0.0% 0.0% 143 ADAMTS1 0.0% 0.0% 0.0% 0.0% 144 ADAMTS10 0.0% 0.0% 0.0% 0.0% 145 ADAMTS12 0.0% 0.0% 0.0% 0.0% 146 ADAMTS14 0.0% 0.0% 0.0% 0.0% 147 ADAMTS16 0.0% 0.0% 0.0% 0.0% 148 ADAMTS17 0.0% 0.0% 0.0% 0.0% 149 ADAMTS18 0.0% 0.0% 0.0% 0.0% 150 ADAMTS19 0.0% 0.0% 0.0% 0.0% 151 ADAMTS2 0.0% 0.0% 0.0% 0.0% 152 ADAMTS20 0.0% 0.0% 0.0% 0.0% 153 ADAMTS4 0.0% 0.0% 0.0% 0.0% 154 ADAMTS6 0.0% 0.0% 0.0% 0.0% 155 ADAMTS8 0.0% 0.0% 0.0% 0.0% 156 ADAMTS9 0.0% 0.0% 0.0% 0.0% 157 ADAMTSL1 0.0% 0.0% 0.0% 0.0% 158 ADAMTSL5 0.0% 0.0% 0.0% 0.0% 159 CD248 0.0% 0.0% 0.0% 0.0% 160 DGCR6 0.0% 0.0% 0.0% 0.0% 161 EGFL6 0.0% 0.0% 0.0% 0.0% 162 EMID1 0.0% 0.0% 0.0% 0.0% 163 FREM1 0.0% 0.0% 0.0% 0.0% 164 FREM2 0.0% 0.0% 0.0% 0.0% 165 RELL2 0.0% 0.0% 0.0% 0.0% 166 SPARCL1 0.0% 0.0% 0.0% 0.0% 167 ACAN 0.0% 0.0% 0.0% 0.0% 168 AMBN 0.0% 0.0% 0.0% 0.0% 169 AMELX 0.0% 0.0% 0.0% 0.0% 170 AMELY 0.0% 0.0% 0.0% 0.0% 171 ASPN 0.0% 0.0% 0.0% 0.0% 172 BGN 0.0% 0.0% 0.0% 0.0% 173 BCAN 0.0% 0.0% 0.0% 0.0% 174 CRTAC1 0.0% 0.0% 0.0% 0.0% 175 CILP2 0.0% 0.0% 0.0% 0.0% 176 CILP 0.0% 0.0% 0.0% 0.0% 177 COMP 0.0% 0.0% 0.0% 0.0% 178 CHL1 0.0% 0.0% 0.0% 0.0% 179 CHI3L1 0.0% 0.0% 0.0% 0.0% 180 CHAD 0.0% 0.0% 0.0% 0.0% 181 C6orf15 0.0% 0.0% 0.0% 0.0% 182 CCDC80 0.0% 0.0% 0.0% 0.0% 183 CTHRC1 0.0% 0.0% 0.0% 0.0% 184 COL1A1 0.0% 0.0% 0.0% 0.0% 185 COL3A1 0.0% 0.0% 0.0% 0.0% 186 COL4A4 0.0% 0.0% 0.0% 0.0% 187 COL4A5 0.0% 0.0% 0.0% 0.0% 188 COL9A1 0.0% 0.0% 0.0% 0.0% 189 COL9A3 0.0% 0.0% 0.0% 0.0% 190 COL5A2 0.0% 0.0% 0.0% 0.0% 191 COL5A3 0.0% 0.0% 0.0% 0.0% 192 COL6A3 0.0% 0.0% 0.0% 0.0% 193 COL8A2 0.0% 0.0% 0.0% 0.0% 194 COL10A1 0.0% 0.0% 0.0% 0.0% 195 COL16A1 0.0% 0.0% 0.0% 0.0% 196 COL18A1 0.0% 0.0% 0.0% 0.0% 197 COL21A1 0.0% 0.0% 0.0% 0.0% 198 COL27A1 0.0% 0.0% 0.0% 0.0% 199 COL28A1 0.0% 0.0% 0.0% 0.0% 200 COLQ 0.0% 0.0% 0.0% 0.0% 201 DMP1 0.0% 0.0% 0.0% 0.0% 202 DSPP 0.0% 0.0% 0.0% 0.0% 203 DPT 0.0% 0.0% 0.0% 0.0% 204 ELN 0.0% 0.0% 0.0% 0.0% 205 EMILIN1 0.0% 0.0% 0.0% 0.0% 206 EMILIN2 0.0% 0.0% 0.0% 0.0% 207 EMILIN3 0.0% 0.0% 0.0% 0.0% 208 ENAM 0.0% 0.0% 0.0% 0.0% 209 EPYC 0.0% 0.0% 0.0% 0.0% 210 ECM2 0.0% 0.0% 0.0% 0.0% 211 FBN1 0.0% 0.0% 0.0% 0.0% 212 FGF1 0.0% 0.0% 0.0% 0.0% 213 FGF9 0.0% 0.0% 0.0% 0.0% 214 FLRT1 0.0% 0.0% 0.0% 0.0% 215 FLRT2 0.0% 0.0% 0.0% 0.0% 216 FLRT3 0.0% 0.0% 0.0% 0.0% 217 FBLN2 0.0% 0.0% 0.0% 0.0% 218 FBLN5 0.0% 0.0% 0.0% 0.0% 219 GPLD1 0.0% 0.0% 0.0% 0.0% 220 GPC2 0.0% 0.0% 0.0% 0.0% 221 GPC3 0.0% 0.0% 0.0% 0.0% 222 GPC5 0.0% 0.0% 0.0% 0.0% 223 GPC6 0.0% 0.0% 0.0% 0.0% 224 HAPLN1 0.0% 0.0% 0.0% 0.0% 225 HAPLN2 0.0% 0.0% 0.0% 0.0% 226 HAPLN3 0.0% 0.0% 0.0% 0.0% 227 HAPLN4 0.0% 0.0% 0.0% 0.0% 228 KERA 0.0% 0.0% 0.0% 0.0% 229 LAMA4 0.0% 0.0% 0.0% 0.0% 230 LAMB4 0.0% 0.0% 0.0% 0.0% 231 LAMC3 0.0% 0.0% 0.0% 0.0% 232 LTBP4 0.0% 0.0% 0.0% 0.0% 233 LOX 0.0% 0.0% 0.0% 0.0% 234 LOXL1 0.0% 0.0% 0.0% 0.0% 235 MATN1 0.0% 0.0% 0.0% 0.0% 236 MATN3 0.0% 0.0% 0.0% 0.0% 237 MMP11 0.0% 0.0% 0.0% 0.0% 238 MMP13 0.0% 0.0% 0.0% 0.0% 239 MMP16 0.0% 0.0% 0.0% 0.0% 240 MMP17 0.0% 0.0% 0.0% 0.0% 241 MMP20 0.0% 0.0% 0.0% 0.0% 242 MMP23A 0.0% 0.0% 0.0% 0.0% 243 MMP26 0.0% 0.0% 0.0% 0.0% 244 MMP27 0.0% 0.0% 0.0% 0.0% 245 MMP28 0.0% 0.0% 0.0% 0.0% 246 MMP8 0.0% 0.0% 0.0% 0.0% 247 MFAP5 0.0% 0.0% 0.0% 0.0% 248 MFAP2 0.0% 0.0% 0.0% 0.0% 249 MFAP4 0.0% 0.0% 0.0% 0.0% 250 MUC4 0.0% 0.0% 0.0% 0.0% 251 MMRN2 0.0% 0.0% 0.0% 0.0% 252 NTN1 0.0% 0.0% 0.0% 0.0% 253 NTN3 0.0% 0.0% 0.0% 0.0% 254 NID1 0.0% 0.0% 0.0% 0.0% 255 NYX 0.0% 0.0% 0.0% 0.0% 256 ODAM 0.0% 0.0% 0.0% 0.0% 257 OPTC 0.0% 0.0% 0.0% 0.0% 258 OMD 0.0% 0.0% 0.0% 0.0% 259 OTOA 0.0% 0.0% 0.0% 0.0% 260 POSTN 0.0% 0.0% 0.0% 0.0% 261 PODN 0.0% 0.0% 0.0% 0.0% 262 PODNL1 0.0% 0.0% 0.0% 0.0% 263 PRSS36 0.0% 0.0% 0.0% 0.0% 264 RPTN 0.0% 0.0% 0.0% 0.0% 265 RBP3 0.0% 0.0% 0.0% 0.0% 266 SPN 0.0% 0.0% 0.0% 0.0% 267 ADAMTS7 0.0% 0.0% 0.0% 0.0% 268 SPOCK2 0.0% 0.0% 0.0% 0.0% 269 SPOCK3 0.0% 0.0% 0.0% 0.0% 270 SPON1 0.0% 0.0% 0.0% 0.0% 271 SFTPA1 0.0% 0.0% 0.0% 0.0% 272 SFTPD 0.0% 0.0% 0.0% 0.0% 273 TECTB 0.0% 0.0% 0.0% 0.0% 274 TNC 0.0% 0.0% 0.0% 0.0% 275 TNN 0.0% 0.0% 0.0% 0.0% 276 TNR 0.0% 0.0% 0.0% 0.0% 277 TNXB 0.0% 0.0% 0.0% 0.0% 278 THBS4 0.0% 0.0% 0.0% 0.0% 279 TFPI2 0.0% 0.0% 0.0% 0.0% 280 TGFB1 0.0% 0.0% 0.0% 0.0% 281 TINAG 0.0% 0.0% 0.0% 0.0% 282 TNFRSF11B 0.0% 0.0% 0.0% 0.0% 283 VEGFA 0.0% 0.0% 0.0% 0.0% 284 VTN 0.0% 0.0% 0.0% 0.0% 285 VWC2 0.0% 0.0% 0.0% 0.0% 286 WNT2 0.0% 0.0% 0.0% 0.0% 287 WNT1 0.0% 0.0% 0.0% 0.0% 288 WNT16 0.0% 0.0% 0.0% 0.0% 289 WNT2B 0.0% 0.0% 0.0% 0.0% 290 WNT3 0.0% 0.0% 0.0% 0.0% 291 WNT3A 0.0% 0.0% 0.0% 0.0% 292 WNT5A 0.0% 0.0% 0.0% 0.0% 293 WNT5B 0.0% 0.0% 0.0% 0.0% 294 WNT6 0.0% 0.0% 0.0% 0.0% 295 WNT7A 0.0% 0.0% 0.0% 0.0% 296 WNT7B 0.0% 0.0% 0.0% 0.0% 297 WNT8A 0.0% 0.0% 0.0% 0.0% 298 WNT8B 0.0% 0.0% 0.0% 0.0% 299 WNT9A 0.0% 0.0% 0.0% 0.0% 300 WNT9B 0.0% 0.0% 0.0% 0.0% 301 ZP1 0.0% 0.0% 0.0% 0.0% 302 ZP4 0.0% 0.0% 0.0% 0.0%

TABLE 10 Most significant Gene Sets Enriched in CTC-pro vs. CTC-c q-value < 0.01 Odds Benjamini Source Term Count Ratio (q-value) GOTERM_BP_FAT GO: 0002495~antigen processing and presentation of 5 59.81 6.97E−04 peptide antigen via MHC class II GOTERM_BP_FAT GO: 0019886~antigen processing and presentation of 5 59.81 6.97E−04 exogenous peptide antigen via MHC class II GOTERM_BP_FAT GO: 0002504~antigen processing and presentation of 5 50.36 7.34E−04 peptide or polysaccharide antigen via MHC class II GOTERM_BP_FAT GO: 0002478~antigen processing and presentation of 5 41.60 1.10E−03 exogenous peptide antigen GOTERM_BP_FAT GO: 0019884~antigen processing and presentation of 5 34.18 1.87E−03 exogenous antigen GOTERM_BP_FAT GO: 0048002~antigen processing and presentation of 5 27.34 3.72E−03 peptide antigen GOTERM_BP_FAT GO: 0001775~cell activation 9 7.00 3.82E−03 GOTERM_BP_FAT GO: 0019882~antigen processing and presentation 6 13.20 7.40E−03

TABLE 11 Most significant Gene Sets Enriched in CTC-plt vs. CTC-c q-value < 0.01 Odds Benjamini Source Term Count Ratio (q-value) GOTERM_BP_FAT GO: 0042060~wound healing 18 7.8 1.86E−07 GOTERM_BP_FAT GO: 0007596~blood coagulation 15 10.4 9.31E−08 GOTERM_BP_FAT GO: 0050817~coagulation 15 10.4 9.31E−08 GOTERM_BP_FAT GO: 0007599~hemostasis 15 10.3 7.59E−08 GOTERM_BP_FAT GO: 0050878~regulation of body 15 8.2 1.30E−06 fluid levels GOTERM_BP_FAT GO: 0030029~actin filament-based 20 5.5 1.14E−06 process GOTERM_BP_FAT GO: 0007010~cytoskeleton 26 3.9 3.95E−06 organization GOTERM_BP_FAT GO: 0030036~actin cytoskeleton 18 5.3 1.11E−05 organization GOTERM_BP_FAT GO: 0009611~response to 26 3.6 1.02E−05 wounding GOTERM_BP_FAT GO: 0007155~cell adhesion 33 2.9 2.86E−05 GOTERM_BP_FAT GO: 0022610~biological adhesion 33 2.8 2.70E−05 GOTERM_BP_FAT GO: 0001775~cell activation 19 3.7 4.70E−04 GOTERM_BP_FAT GO: 0030168~platelet activation 6 18.2 1.68E−03 GOTERM_BP_FAT GO: 0007229~integrin-mediated 10 6.4 2.95E−03 signaling pathway GOTERM_BP_FAT GO: 0016192~vesicle-mediated 25 2.6 3.81E−03 transport MSigDBv3.1 CGP GNATENKO PLATELET SIGNATURE 20 55.1 3.91E−24 MSigDBv3.1 CGP TENEDINI MEGAKARYOCYTE 14 15.3 1.35E−11 MARKERS MSigDBv3.1 REACTOME FACTORS INVOLVED IN 6 2.9 2.25E−02 CP: REACTOME MEGAKARYOCYTE DEVELOPMENT AND PLATELET PRODUCTION

TABLE 12 Significantly Expressed Genes by Rank Product (FDR <0.01) Primary CTC-c vs Tumor vs Primary CTC-c Tumor Enriched CTC-plt vs Count Enriched Gene Gene CTC-c CTC-pro vs CTC-c 1 Upk3b Tff2 Clec1b kg:uc007pge.1 2 Ier2 Wfdc2 AU023871 kg:uc007pgd.1 3 Egr1 Lamb3 Alox12 kg:uc007pgf.1 4 Nkain4 Lad1 Itga2b kg:uc007pgg.1 5 Igfbp5 Dmbt1 Ppbp Igj 6 Slc6a4 Npy Gng11 kg:uc012enb.1 7 Klf4 Pmepa1 Vwf 2010001M09Rik 8 Tmem221 Kcnn4 Pf4 kg:uc009cfw.1 9 Arl4d Serinc2 Fcer1g kg:uc007pgi.1 10 Lrrn4 5730559C18Rik Tmem40 kg:uc007pgh.1 11 Cldn15 Muc1 Hba-a2 kg:uc007yos.1 12 Gpm6a Chi3l3 Stom Coro1a 13 Atf3 Pglyrp1 Beta-s Pou2af1 14 Ptma Arl4c Plek kg:uc011yvj.1 15 Slc9a3r1 Spp1 Srgn Glipr1 16 Fos Col15a1 Myl9 Cd52 17 Tmem119 C1qb Cd84 Cd79b 18 Ptgis Tnnt2 F5 Sec11c 19 Dcn Gkn3 Treml1 Tnfrsf17 20 Gbp2 Onecut2 Hbb-b1 Krr1 21 Dmkn Mmp7 Itgb3 Gmfg 22 Sdc4 Cd74 Gp9 Ccr9 23 Ildr2 Ctss Mpl Pycard 24 Akap2 Lamc2 Ctla2a Derl3 25 Gfpt2 Olfml3 Tubb1 Rac2 26 Klf6 Lgals4 Mylk Srgn 27 Btg2 Lcn2 F13a1 Cytip 28 Myl7 Ly6a Slamf1 Edem2 29 Igfbp6 Pak1 Rgs10 Itgb7 30 Gpr133 Capn5 Mkrn1 Lsp1 31 Oasl2 Ptprn Laptm5 Lcp1 32 Pfn1 Reg3b 1810058l24Rik Cyfip2 33 Cap1 Fmnl3 Itgb2 Nans 34 Nfkbia Sdc1 Slc2a3 Slamf7 35 Malat1 Prom1 Pcmt1 Ell2 36 Rarres2 Ankrd50 Gp5 H2-Eb1 37 Rspo1 Ccl6 Ube2o Creld2 38 Espn Slc4a11 5430417L22Rik Cd74 39 Klf9 Oraov1 Ptpn18 Blnk 40 Zbtb7c Aldh1l1 Lat Fmnl1 41 Brd2 Slc20a1 Fermt3 Snrnp70 42 Olfr1033 Cldn7 Nrgn Sec61b 43 Wt1 Acsbg1 Mrvi1 Edem1 44 Esam Las1l Lyz2 Tspan13 45 kg:uc009igb.1 C1qc Epb4.1 Psmb8 46 Tmem151a Lama5 Rasgrp2 Pim1 47 Mgll Mgat4a Treml2 Sept1 48 Csrnp1 Cldn2 Hist1h4i Cd48 49 Cd9 Mcpt2 March2 Sub1 50 Gjb5 Fxyd3 Ltbp1 Lims1 51 Lrrc61 Il4ra Nptn Ncoa2 52 Wasf2 Itga5 Abtb1 Ctnnbl1 53 Pdpn Porcn Ctla2b Fdps 54 kg:uc009ogv.1 Mast3 Prkab2 Ube2j1 55 Sdpr Scara3 Arhgdib Mettl1 56 Gpr64 Atox1 Alas2 Lax1 57 Flnc Arrdc1 Odc1 Rilpl2 58 Add3 Mmp2 Ptpn11 Ctse 59 Gata6 Saa3 Dhcr24 Glrx 60 Wfdc1 Serpinf1 Mfsd2b Fut8 61 A130040M12Rik Sox11 Gp1bb Al662270 62 Ankrd12 Prpsap1 Rbpms2 Gramd3 63 Adamtsl1 Mcpt1 Fyb Il2rg 64 C2 Mfge8 Smox Rasgrp3 65 Prss23 Col18a1 P2rx1 Impdh1 66 Ube2v1 Lyz2 Otud7b Plek 67 Cryab C1qa kg:uc007ttx.1 Ints5 68 Pkhd1l1 Acp5 Samd14 Blmh 69 Rtn1 Angptl4 Clca1 Dnmt1 70 Birc6 Ccnd1 kg:uc007tty.1 Galk1 71 Xdh Asl Gpr56 kg:uc007hxv.1 72 Cd34 Ctxn1 Sh3bgrl2 Ccdc88b 73 Rab6b Pgs1 Pttg1ip Selplg 74 Dusp1 Anapc2 Nomo1 Sar1b 75 Clic4 Cp Gnaz Lat2 76 C3 Gpx3 Mmrn1 Slc16a6 77 Rhob Lama3 Gp1ba Mki67 78 Mir3064 Rbp1 Sh3bgrl3 Dnajc3 79 Thbd Cotl1 Slc24a3 H2-Ab1 80 Dpysl2 Nek6 Sord Ndufs6 81 Cobl Cpxm1 Nfe2 Actr3 82 Npr1 Sfrp1 Tuba4a Etnk1 83 Dnajb9 Ttr Zyx Herpud1 84 Arhgap29 Gsto1 Cnn2 Ptpn7 85 Cav1 Npepl1 Itgb5 Ctss 86 Gbp7 Usmg5 Gata1 Cs 87 Hes1 Polr2l Hist1h1c Fbxw7 88 Gm16897 Sphk1 Tbxas1 Ppp2r5c 89 Ppp1r12a Asxl1 Ptplad2 Znrd1 90 Sv2a Ctsh Bpgm Rfc2 91 Ang Egfl7 Pdlim7 Preb 92 Aldh1a2 C1qtnf6 Mmd Fcer1g 93 Cryl1 Rras G6b Dnajb11 94 Kank1 Lgi4 kg:uc009duo.1 Slc35b1 95 2210403K04Rik Hmga2 Lyz1 Sin3b 96 kg:uc009okn.1 Cep250 Tacc1 Nktr 97 Osr1 B4galt3 Dap 98 kg:uc008ewj.2 Tmem223 Mast2 99 kg:uc009tuw.1 Ltbp2 Atp2a3 100 Gadd45b Tnfrsf23 Snca 101 Ablim3 Col7a1 Stx11 102 Clec3b Ggct C030046I01Rik 103 Usp25 Rab25 Trpt1 104 Sntb2 Nedd8 Tsc22d1 105 Rock2 9430023L20Rik Prkar2b 106 Col14a1 Arl2 Cd9 107 Cd200 Wbp1 Pgm2l1 108 kg:uc008ehr.1 H2-Ab1 Gp6 109 Atp2b1 Preb Pde5a 110 Exoc4 Sgsm3 Itga6 111 Abcb1b Sfn Itgal 112 Nrgn Prrx2 Edem1 113 kg:uc009cvm.1 Ptprk Isg20 114 Ncoa4 Reg1 Cdc42ep5 115 Ndufa4 Sdcbp2 Nipal3 116 Upk1b Pcbd1 Ccdc92 117 Jun Slc25a1 Sort1 118 Syne2 Vamp5 Ly6g6c 119 kg:uc007bvx.1 Crlf1 Ubash3b 120 Ap4e1 Avil Inf2 121 Spock2 2700094K13Rik Asap1 122 Efemp1 Ctse Sec11c 123 Prpf40a Penk Gas2l1 124 Tspan5 Tmc4 Parvb 125 Lgals7 Dhrs3 Tmsb4x 126 Kif5b Ap1s1 kg:uc007xrw.1 127 Psip1 Arl6ip4 Nudt3 128 kg:uc008oki.1 9430008C03Rik Bcl2l1 129 1810014B01Rik Fcer1g B230312A22Rik 130 Ptges3 Uqcr11 Cnp 131 Limch1 Nhp2 Plp1 132 Bicd1 Plbd2 Cnst 133 Rdx Capg Rgs18 134 Pcdh15 Pnpla6 Lsm12 135 Foxn3 Ppdpf Alox5ap 136 Morf4l2 Hgfac Ppif 137 Ppp1r15a Apoe Spnb1 138 Cdc42ep3 Fam40a Ormdl3 139 Pard3b Lyz1 Hpse 140 Bicc1 2200002D01Rik Srxn1 141 Amhr2 Laptm5 2010002N04Rik 142 Gucy1a3 Qars Hist1h2bc 143 Psmb2 Tmx2 Cyba 144 Mapkapk3 Fkbp4 Chst12 145 Ube2l6 Plin2 kg:uc009sps.1 146 kg:uc007pff.1 Fcgr3 Max 147 kg:uc007ctp.1 Gkn1 Was 148 Nedd4 Snhg1 Isca1 149 Plxna4 Lsp1 Pdzk1ip1 150 2010107G12Rik Gm20605 Lyn 151 Ifngr1 Ly6c1 Mob3a 152 Bcam Aim1 H2-T24 153 Ccnl1 2310007B03Rik Slc44a1 154 Hoxa5 Tgfbi Derl1 155 Fhl1 Tsta3 Gclm 156 1810041L15Rik Pafah1b3 Fech 157 2900002K06Rik Chid1 Ywhah 158 Hspb1 Smox Igtp 159 Podn 1500012F01Rik Myl6 160 Fam63b Tspan4 Thbs1 161 Hsp90b1 Agrn Tln1 162 Dpp4 Cfp kg:uc009apq.1 163 Gas1 Cdh1 Bcap31 164 kg:uc007zak.1 Rasgrf1 Ilk 165 Zc3h13 Nxf1 Epha1 166 Sox6 Pdrg1 2810453I06Rik 167 Arid4a Polr2j Rnf19b 168 Tnxb Suds3 Gsn 169 Tsix D0H4S114 Flna 170 Scd1 Ccl9 Arrb1 171 Jund Neat1 kg:uc007pum.1 172 Crls1 Ccdc12 Mbnl1 173 1110003E01Rik Prr24 Ccnd3 174 Rnase4 Impdh1 Pdlim1 175 Arhgef12 Card10 Ctse 176 Irf7 Cpsf1 Tspan17 177 Bbx Sema4g Gpx4 178 Sema5a Hes6 Bnip3l 179 Mau2 C130074G19Rik P2ry12 180 Abi3bp Ctrb1 kg:uc009vev.1 181 Dag1 Rnaseh2a Prkab1 182 Cyp2s1 Golm1 F2rl2 183 Sfrs18 Ctsz Stk4 184 Hspb8 Cyb561 Fhl1 185 Cnot6l Ndufs8 Rnf10 186 Twsg1 Atp6ap1 Rasa3 187 Gpc3 Srd5a1 Taldo1 188 Lrrn4cl Carkd Bysl 189 Cdh3 Cd24a Esd 190 Cyr61 Eng Aldh2 191 Cyp2d22 Tcirg1 Rhog 192 Hist1h1c Slc9a3r2 kg:uc009ecr.1 193 Aplp1 0910001L09Rik Cald1 194 Tbl1x Cox5b Wbp2 195 Pcm1 Adipor2 Ptprj 196 Ifi204 Scarf2 Tpm4 197 Nfix Myo7a Mxi1 198 Flrt2 Ppap2c Ly6g6f 199 Heg1 Pea15a Sla 200 Il6ra Sh3pxd2b Slpi 201 Ralbp1 H19 Bicd2 202 Rhoj Tpd52 Clu 203 Ktn1 2610203C20Rik Mtmr14 204 Arl6ip5 Naa10 Abca7 205 Crebbp Fermt1 Ppp1r18 206 Ppig Sap30l Kif2a 207 Akap13 Bgn Prdx6 208 Rab7 Timm13 kg:uc009ize.1 209 Plxdc2 Krt20 Calm3 210 Aldh1a1 Itga3 Dhrs1 211 Bnc2 Pfkl Cfl1 212 Slc4a4 Agpat6 Glipr2 213 Tbx18 Mrpl11 Slc25a37 214 Zbtb16 Ramp1 Atox1 215 Arid4b Hmga1 BC057079 216 Enpp2 Gpx2 Pla2g16 217 Ptplad2 0610012G03Rik Rnf144b 218 Akr1b3 9130017N09Rik Stk16 219 Gm6644 Cygb Rsad2 220 Arf5 Tmprss4 Paip2 221 Chi3l1 Paox Capzb 222 Gpr116 Endod1 Ppp1r12c 223 Cd82 Cndp2 4930412F15Rik 224 Srrm1 Suv39h1 Ninj1 225 Fmo2 Cog4 2510009E07Rik 226 Tgfb1i1 Trim27 kg:uc007vsr.1 227 Qrich1 Cyhr1 Pygb 228 Nfia Trmt1 Tlk1 229 Pmp22 Zfyve19 Myct1 230 Cdh11 Esrp1 Rnasek 231 Arid5b kg:uc008oow.1 Ctsd 232 Rbm3 Dync1h1 0610010K14Rik 233 Prelp Tab1 Bcas3 234 kg:uc007qse.1 Pla2g6 Atpif1 235 Ddx3x Timp1 Serf2 236 Sulf1 Eif3f Becn1 237 Spnb2 Abhd11 Tspan9 238 Tspan31 Pmm2 Acer2 239 Prr13 Tyrobp Vdac3 240 Ppp1cb Farsb kg:uc008kbg.1 241 Fbln1 Plod3 Oaz2 242 Gm6548 Abtb1 Serpine2 243 Uap1 Brf1 Ccdc90a 244 Mpdz Tnk2 Ndufa1 245 Sat1 Rfc2 Tssc1 246 Stim1 Stxbp2 Mboat7 247 Mll3 Pdlim7 Cd44 248 Slurp1 A430105I19Rik Cxx1c 249 Cd81 Vill Ecm1 250 Emp2 Bmp1 Mff 251 Trpm7 Mpzl1 Ptpn12 252 Crym Thy1 Mgmt 253 Enpp4 Stab1 Cox4i1 254 Raly Aldh16a1 Tollip 255 Celf2 Eif4ebp3 Cds2 256 Ap3s1 Itpripl2 Ybx1 257 C1s Mrpl52 Gypc 258 Frmd4b 2310002L13Rik Dgkd 259 Nr4a1 Mcm6 Pecam1 260 Acin1 Kcnk1 Ftl2 261 Plod2 Pmf1 Nt5c3 262 Id1 Cuta 1700037H04Rik 263 Creg1 Nt5dc2 Cd151 264 Zfp318 Rmnd5b Lpin2 265 Tmem140 Araf 6430548M08Rik 266 Mras Wwp2 Pon2 267 Vwa5a Lamb1 Ndufa3 268 Esyt3 Kcne3 6330578E17Rik 269 Hexb Uqcrq Mfap3l 270 Nckap1 Gps1 Mink1 271 Nipal3 Rexo4 Ston2 272 Ubxn4 Coro1c Rac2 273 Zfp36 Hras1 Fyn 274 Hnrnpl Spint1 Serinc3 275 C1ra Cblc Maged2 276 Nnmt Fhod1 Ap2m1 277 Mut Atp13a1 Pacsin2 278 kg:uc008jup.1 Man2c1 Ftl1 279 Pnrc1 Vsig2 Adipor1 280 Usp8 Bpgm kg:uc009qdo.1 281 Pgcp Bap1 Snap23 282 Junb Smpd2 Tagln2 283 C1rl Ubqln4 Cox6c 284 Slc6a6 Sirt7 Creg1 285 kg:uc008znh.1 Krt23 Bsg 286 Aqp1 D8Ertd738e Cmtm6 287 Myh10 Mapk13 Cntd1 288 Slc43a3 kg:uc008bcq.1 Plekho2 289 Spint2 Polr2g Arrb2 290 Hnrnph1 Ndufs2 Pard3b 291 Arhgap28 Dad1 Mlec 292 Cfh Wnt7b Taf10 293 Brd4 Fam20c Gabarapl2 294 Fndc1 Cxxc5 Bag1 295 Star Polr2f Galnt2 296 Nfkbiz Ltf Hk1 297 Arsb 2210407C18Rik Fbxo9 298 Rnd3 Cdipt kg:uc009izd.1 299 Stard5 Glrx5 Pnpo 300 Thbs1 Gemin7 Fam46c 301 kg:uc008wkn.1 Man1b1 Pkm 302 Slc26a3 Heatr7a Ap1b1 303 Phip Arid5a Rap1b 304 Usp2 Sumo3 Itgb1 305 Golgb1 Srm St7 306 Rock1 Plscr3 Smap1 307 Rgma 2210010C17Rik Rabgap1l 308 Actg1 Fam102a Tmbim4 309 BC013529 Dlst H3f3a 310 kg:uc007zwh.1 Vps37c Frmd8 311 3110062M04Rik Ngfrap1 Nlrx1 312 Cast Pold4 Oaz1 313 Mob3c Grcc10 Fam125b 314 Slc16a1 Wnt7a Hexa 315 Fam117a 2010111l01Rik Tspo 316 Pdia3 Pxdn Dcaf12 317 Trim8 Coasy Nav1 318 kg:uc009mng.1 Dctn1 Cd24a 319 eg:245190:chr7:m Ncor2 Uqcr11 320 Sbsn Postn Wipf1 321 Serpinb6b Col4a2 F10 322 Daglb Cib1 Erlec1 323 Smarca2 Tbc1d13 Map2k3 324 Mef2c Ccnl2 Stk24 325 Prrc2c Dcakd Ldlrap1 326 BC005537 Cdc34 Ehd4 327 Hsp90ab1 Atp6v0b Atp6v1f 328 Snrnp70 Abhd12 Gnas 329 Ppl Flot2 Arhgap18 330 Serpinh1 Sla2 Arhgap10 331 Sorbs3 Rhbdf1 Pitpnm1 332 Golga4 Cdh17 S100a1 333 Acbd3 Psmb5 Bin1 334 Hook3 Serf1 Ttyh3 335 Map3k3 Slc15a3 Selp 336 Rhou Sftpd Trappc9 337 Smc2 Pop5 Aes 338 C1d Nudc Taok3 339 kg:uc008dzh.1 Sh2d5 Zfand3 340 Psmd7 kg:uc007fwp.1 Stim1 341 Dab2 Mrpl37 Rnf114 342 Cep164 Rin1 Sep15 343 Crim1 Podxl kg:uc012hdk.1 344 Rtf1 Paqr5 Lgals9 345 Fxyd1 Sepx1 Cox6b1 346 H2-D1 Agr2 Riok3 347 Zfp704 Bax Slc38a10 348 Mtap1a Rxrb Rtn3 349 Ascc3 Tes B3gat2 350 Med13l Hdac6 Ccndbp1 351 Jup 1110008F13Rik Rsu1 352 Nid2 Mpnd kg:uc007upr.1 353 Kdr Gmppa Itm2b 354 Ifnar2 Gramd1a St3gal1 355 5430435G22Rik Wars Sec61g 356 Col4a6 Mtap Ptpn1 357 Il17re C1qtnf5 kg:uc012bhf.1 358 Gbp3 Mrpl28 B2m 359 Slc39a8 Mfrp Rasgrp3 360 Cfl2 Kars Memo1 361 Slc38a1 Lbp Slc39a4 362 Cuedc1 Plxnb1 Sdcbp 363 Fgf1 2700081O15Rik Tspan14 364 Gas6 Mrps24 Ubl7 365 Cldn25 Klc4 Nras 366 Sorbs1 Dctn3 Ssx2ip 367 Hspa12a Kcnq1 kg:uc007zbz.1 368 kg:uc007zts.1 Smurf1 Wbp1 369 Slc1a5 Fam162a 1110003E01Rik 370 Nr3c1 Hip1r Clip2 371 Adamts5 kg:uc007hyr.2 Gapdh 372 Gpcpd1 Gys1 Gm6578 373 Dpysl3 Sac3d1 Actn1 374 Colec12 Ndufs6 St3gal2 375 Pdcd6ip Rgl2 3110001D03Rik 376 Dst Atp5g1 Ctsz 377 Ifit3 Itgb4 kg:uc007vdl.1 378 Chst4 Sars Fam73a 379 Xist 2310003F16Rik Vcl 380 Ifi27l2a Nhp2l1 Lims1 381 Fkbp5 D19Wsu162e Lars2 382 Agap1 Cd320 Birc2 383 Ankrd11 Pigq Lamp2 384 kg:uc007qca.1 Chd3 Rasl10a 385 Syt11 Zdhhc4 Mif 386 Ptrf Eif3l Rab10 387 Krcc1 St8sia3 Pabpc1 388 Zfp488 Rcan3 Wwp2 389 Lama4 Meg3 Nqo2 390 Aebp1 Nudt4 kg:uc007fte.1 391 Fam134b Gss Plxna4 392 Tppp3 Pih1d1 Gm1821 393 Maf Limd2 Gadd45a 394 Peli1 Ap1s2 Slc25a39 395 Zfp353 BC056474 kg:uc009pet.1 396 Cdon Mms19 Ubb 397 Sarnp Clip2 Ppp1r2 398 Atxn7l3b 2310016M24Rik Rab27b 399 Pef1 Itpa Cap1 400 App Slc25a10 Jarid2 401 Mtdh Fibp Rnf11 402 Lrrc20 Higd2a Tmem50b 403 Btbd2 Snrpd2 Myh9 404 Gnb2 Eri3 Tmem128 405 Pigt Nbeal2 Stradb 406 Efna5 Trim28 Cela1 407 Tm4sf1 S100a4 Ndrg2 408 Coq10b Ivns1abp Dhrs3 409 Eif2s3x Ppp1r18 Hipk1 410 Cmah Efemp2 Atg9a 411 Sf3b1 Med22 412 Eea1 Nelf 413 Slpi 2810428I15Rik 414 Tmod3 D2Wsu81e 415 Ppp3ca Trappc6a 416 Tceal8 Trappc2l 417 Anp32a Antxr2 418 Actb Rab11fip5 419 Ddx5 Ldhd 420 Cobll1 Npnt 421 Cish Acrbp 422 Nod1 Pafah1b2 423 Psd Angptl2 424 Gm10052 Fzr1 425 Lims2 Aaas 426 Stra6 Eif2b2 427 kg:uc007bgn.1 1190003J15Rik 428 Plxdc1 5730403B10Rik 429 Nfe2l1 Adamts13 430 Smpd3 Eif3b 431 Bcl10 Znrf1 432 Ilf3 Pkp3 433 Fam76a Lemd2 434 Cybrd1 Rab34 435 Gm3893 Mpv17l2 436 Siae Cdkn2b 437 Ssh2 Snrpe 438 Nfic Gm14005 439 Btf3 Prdx4 440 Sp100 Xab2 441 Ndn Dpp3 442 Matr3 Tyms 443 Gm13251 Leprotl1 444 Arhgap5 Uqcr10 445 Zbtb4 Cdk5rap3 446 Pgrmc1 Gorasp2 447 4930402H24Rik Wbp7 448 Bptf Sort1 449 Dusp3 Ddx41 450 Pla2g4a Cct3 451 Brp44l Mrps33 452 Oxct1 Frmd8 453 Stk40 1110049F12Rik 454 Ddr1 Fscn1 455 Ifi205 Ndufa2 456 Col3a1 Dpcd 457 Nipbl Unc13a 458 Plk1s1 Eif1ad 459 Bdp1 Sgta 460 Smc3 Chaf1a 461 Ifitm3 Plxna1 462 Ndst1 Hspa9 463 Zbed6 1110014N23Rik 464 Rest Cd99l2 465 kg:uc007vnc.1 Snrpa 466 Ccdc88a Mcm7 467 Stat3 Tars2 468 Arf2 Gon4l 469 Trib1 Stk38 470 Gcap14 C1qtnf1 471 Tbc1d15 Tbrg4 472 Igf1r Tmem132a 473 Ppbp Cox6c 474 kg:uc008tky.1 Alcam 475 Rab1b Phka2 476 Krt14 Trim3 477 Med21 Ppp1r14b 478 Gja1 Gpaa1 479 Klf10 Ctps2 480 Id2 Ptpn23 481 Mfap1a Endog 482 Ogn Mrto4 483 Gpc4 Mrps6 484 Bst2 Pvr 485 Dtx2 Phgdh 486 Wac Itpr3 487 Kpna3 Polr2e 488 Kcnab1 Sec16a 489 Orai3 Mdp1 490 Gcsh Fbf1 491 Wdr92 Mcpt8 492 Olfr613 Rps6ka4 493 Tcf7l1 Mical1 494 Tgfb2 Mrpl34 495 Il16 Agpat3 496 Manf 2310044H10Rik 497 Mgst1 Myo9b 498 kg:uc008tkz.1 Ndufb10 499 Creb3l1 Apex1 500 Txndc5 Elk3 501 Klf2 Cpsf3l 502 Slu7 Tnk1 503 Ttc28 Pmvk 504 1110002B05Rik Ppp1r16a 505 Zcchc11 Arhgef5 506 Ptp4a2 Lonp1 507 Pbx1 Pla2g7 508 Clcn3 Pip5k1c 509 Tmco7 Inf2 510 Lrrc58 Pgk1 511 Eif3a Parp6 512 Cldn10 Urm1 513 H2-Q6 Mad2l2 514 Ccdc80 Ing4 515 kg:uc009iln.1 Rbck1 516 Rab5c Cant1 517 Tsc22d3 Sgpl1 518 Tm4sf5 Ehbp1l1 519 Hmgb1 Runx1 520 Sec62 Slc27a4 521 Maoa Ndufa7 522 Clec1b Mcm3ap 523 Mphosph8 1110008P14Rik 524 Oat Rassf7 525 Ncor1 Ptpmt1 526 Cyb5 Arfgap1 527 Trafd1 Sec61a1 528 Rpp25 Rps6ka1 529 kg:uc007ded.1 Ints1 530 2610101N10Rik Tpcn1 531 Il6st Iffo2 532 Evpl Trim44 533 Psmd11 kg:uc012ctw.1 534 Dync1i2 Golga2 535 Lars2 Msto1 536 Pdia4 Ppp6r3 537 Cd55 Trmt2a 538 Amfr Appl2 539 Zcchc3 Sparcl1 540 Herpud2 Rapgef1 541 Txnrd1 Zfpl1 542 Vat1 Psmc4 543 Diap1 Mosc2 544 Tmed2 Fam101b 545 Arf3 1500010J02Rik 546 Arap2 Ccdc124 547 St3gal1 Ptges 548 Man1a Fam189b 549 Rgs10 Th1l 550 Tmsb4x Kctd2 551 Uba7 Olfr1372-ps1 552 C4b Hexa 553 Tmem98 Anapc5 554 Lpar2 Serpina3n 555 Gabarapl1 1810046J19Rik 556 Cmtm7 Tmem167 557 Spon2 Gm11428 558 Smarca5 Gcn1l1 559 Mxd4 Kansl3 560 Smc4 Fasn 561 Thsd4 Slc50a1 562 Gsr Smad3 563 Ptprd Trip6 564 Clip1 Atp6v1e1 565 Cln8 Chchd5 566 Rbm27 Adssl1 567 Zmat1 Nes 568 Smc6 Ap1b1 569 B2m Fcgrt 570 Irf2bp2 Ltbp3 571 Ppap2a Csf2rb 572 Zfhx4 Ssna1 573 Tob2 Mrps16 574 Rabgap1l Cyba 575 Nfkb2 Cyth2 576 Nfyc Igf2 577 Ube2d1 Pisd-ps1 578 Creb5 Atp13a2 579 Opa3 Mlph 580 Csnk1a1 Cyp4f16 581 Fam84b 2010107E04Rik 582 Ddr2 Gas5 583 Usp54 Eif3k 584 Akt2 Fam149a 585 Strn3 Mif 586 Hnrnpm B230312A22Rik 587 eg:497210:chr14:m Ppp1r12c 588 Tpt1 Tfip11 589 Naa25 Tex10 590 Eef1a1 Slc16a3 591 Parp4 Stk16 592 Msn Epn1 593 Zbtb20 Noc4l 594 Fermt2 Rcc2 595 Bod1l Rgs12 596 Sltm Shkbp1 597 Dapk1 Got2 598 Hnrnpr Plek2 599 Baz2a Lilrb3 600 Rnf167 Ndufb5 601 Mapk1 Tesk1 602 eg:320169:chr9:p Rab24 603 4930523C07Rik Atp5j2 604 Nf1 Commd9 605 Fam53b Rtkn 606 Faim2 Prpf19 607 Tgm2 6720401G13Rik 608 Calm2 Ppa1 609 AI848100 Pgp 610 Slc10a3 Hps1 611 Ogdh Puf60 612 Arl3 Mdm2 613 Timp2 kg:uc012cgd.1 614 Atxn2 kg:uc009uim.1 615 Mll1 Pyy 616 Ces2g Zfp358 617 Mat2a Timm8b 618 Esf1 Ddx39 619 Hsp90aa1 Pgm2 620 Zfp385a kg:uc008gbp.1 621 Zfp672 Sipa1 622 Csda Mgat1 623 Pf4 Tmem208 624 Arsa Ruvbl2 625 F11r 8430410A17Rik 626 C4a Bad 627 Kpna1 Pfdn5 628 Rbbp8 Eme1 629 Oxnad1 kg:uc009mzj.1 630 Rb1cc1 Igf1 631 Setd2 Prkag1 632 Kif1b kg:uc009sua.1 633 2510002D24Rik Uap1l1 634 Cep57 Trappc4 635 Chd2 Bola2 636 Serinc5 Usp5 637 Marcksl1 Ear2 638 Shfm1 Cars 639 Bbs4 1810027O10Rik 640 Impad1 Amdhd2 641 Tbcel Phb 642 Kdelr1 Kcmf1 643 Ninl Lsmd1 644 Sytl1 Sec11c 645 Tpm3 Pcbp4 646 Rbbp6 Mepce 647 Lman1 Tpd52l2 648 Ankrd17 Trf 649 Naga Hsd17b11 650 Rbpms Pilra 651 Magt1 Atn1 652 Tfdp2 Pgf 653 Gem Nxn 654 Pde4dip Inpp5k 655 Mrgprf Actr1a 656 kg:uc008ajk.1 Cd68 657 Itch Eef1g 658 Elf1 Fbn1 659 Meis2 Hint1 660 Arid1a March5 661 Serping1 Usp48 662 Slc27a3 Hnf1b 663 Thoc2 Gga3 664 Gsta3 Drosha 665 Hnrnph2 Ubp1 666 Socs3 Pkn3 667 Armcx3 Tmem192 668 Siah1a Prpf31 669 kg:uc009ize.1 Hspd1 670 Irs2 Otub1 671 Mettl7a1 Mrpl20 672 Ppfibp2 Tead2 673 Blvrb Phpt1 674 Yipf5 Neu1 675 Plat Pygo2 676 Gm6578 Myeov2 677 Mat2b Cdk5 678 Tmpo Ndor1 679 Metap2 Rbp4 680 Zfp277 Psat1 681 Wls Mrpl41 682 Mesdc1 Snrpg 683 kg:uc009acs.1 Acot7 684 Col1a2 Vars 685 Csf1 Nono 686 Sulf2 Gtf2i 687 Ifrd1 Traf3 688 Wrnip1 Ppp2r4 689 Flii Actg2 690 2810474O19Rik Pi4k2a 691 Sep15 Slc35b2 692 2310030G06Rik Ubqln1 693 Cmtm3 Ppox 694 Mylip Bud31 695 Slc8a1 Man2b1 696 Btbd7 Nat15 697 Hdac5 Spon1 698 Zfand6 Cyc1 699 Tapbp Mpeg1 700 Keap1 Nsun2 701 Ube2n Rab4a 702 Ssr3 Mtmr11 703 H3f3a BC004004 704 Myst4 B4galnt1 705 G3bp1 Atp5k 706 Ugdh Lin37 707 Lamp2 D330041H03Rik 708 Zrsr1 Tbc1d17 709 Pim1 March6 710 Gm9199 2410015M20Rik 711 Supt16h 1810013D10Rik 712 Ano6 Eif2s1 713 Soat1 Traf7 714 Eci1 Rpl36al 715 Plce1 Psenen 716 Atg3 Aip 717 Bnc1 Cmas 718 Pik3c2a Rpia 719 Pqlc3 Ncbp1 720 Thrap3 Mea1 721 Irak4 Timm50 722 Kdm6b Ear12 723 Apol9a Fkbp1a 724 Wnt4 Commd4 725 1500003O03Rik Col5a3 726 Phf3 Fblim1 727 1110004F10Rik Cwh43 728 Kansl1 Arl2bp 729 Fth1 Mrpl46 730 Tmem50a Tcn2 731 Utp20 Add2 732 Smad4 Specc1l 733 Stmn2 Ppcs 734 Gstm1 Vrk3 735 Senp6 Trim25 736 Gda Nfatc1 737 Nucks1 Rap1gap 738 Ints10 Hsd17b12 739 Syne1 Epas1 740 Itga6 Ddx1 741 Acad9 Prdx6 742 Maged1 Mmp24 743 Spen Ndufb9 744 Chd1 Phf23 745 Taf3 Rpa2 746 Ptgs1 5031439G07Rik 747 Sparc Rrp7a 748 R74862 Arfip2 749 B230120H23Rik Efna1 750 Tmem234 Agps 751 Ryk Sephs1 752 Dlgap4 Apoc2 753 Atp1b1 Mrps27 754 Parp14 Snn 755 Tgfbr2 Serinc3 756 Ccdc90a Pdcd5 757 Ncoa1 AA986860 758 Pppde1 Pitpna 759 Luc7l3 Vac14 760 Prg4 2810025M15Rik 761 Rab11fip1 Def8 762 Plk2 Hilpda 763 Ifi35 Eif6 764 Pdap1 Brd7 765 Cd248 Fes 766 Sesn1 Sbf1 767 Ecd Ak2 768 Ap1s3 1810035L17Rik 769 H2-K1 Lime1 770 Spag9 Hspe1 771 Tshz1 Csrp2bp 772 Dennd5a Uba5 773 Stag1 Gsta4 774 Gpx8 2900092E17Rik 775 Sod3 776 BC005561 777 kg:uc009vev.1 778 Ywhaz 779 Ganab 780 Rras2 781 Dusp14 782 kg:uc012hdk.1 783 Nr1d1 784 Wwc2 785 Ubxn2a 786 Iqsec1 787 kg:uc007vsr.1 788 Cfl1 789 Csrp1 790 Smchd1 791 Myl12a 792 Ubqln2 793 Tmcc3 794 Kdm5a 795 Rbm25 796 Wdr26 797 Vim 798 Arpc2 799 Calm1 800 Dnaja2 801 Shc1 802 Vps13a 803 Klf7 804 1810074P20Rik 805 BC003331 806 Itpr2 807 Jmjd1c 808 Pcdhgb5 809 Tubb2a 810 Ehd2 811 Ift74 812 Per1 813 Pitpnm2 814 Gstm4 815 Dnmt1 816 Tmco1 817 Lass4 818 Ptprf 819 Sirt2 820 Gfm2 821 Taf7 822 Spop 823 Zzef1 824 Ccdc34 825 Zfp281 826 Tuba1a 827 Ccdc109b 828 Cdk13 829 Dhx15 830 Src 831 Braf 832 Mapre2 833 Anxa7 834 Sept9 835 Alox12 836 Pknox1 837 2610034B18Rik 838 Topors 839 Phf21a 840 Qser1 841 Tirap 842 Fas 843 Lass2 844 6330406l15Rik 845 Parvb 846 Atp1a1 847 Mtmr6 848 Cd109 849 Dnajc1 850 Hp1bp3 851 1600029D21Rik 852 Ttc38 853 Mfhas1 854 Filip1l 855 Zfp148 856 Nkd1 857 Usp16 858 Tlr2 859 Zc3h18 860 Stk10 861 Ltbp4 862 Hdac3 863 Efhd2 864 Prkar2a 865 Atp6v1a 866 Sf3b4 867 Gprc5b 868 Clip3 869 Mettl2 870 Secisbp2 871 Fmod 872 kg:uc009lxf.1 873 Elovl6 874 Bzw1 875 Etfa 876 Hspa2 877 kg:uc007won.1 878 Rnf20 879 880 881 882 883 884 885 886 887 888 889 890 891 892 893 894 895 896 897 898 899 900 901 902 903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 921 922 923 924 925 926 927 Primary WBC vs CTC-c Tumor Primary vs WBC vs Count vs WBC Tumor WBC CTC  1 Wfdc2 Ppbp Olfr1033 Beta-s  2 Spp1 Alas2 Crip1 Alas2  3 Cct3 Nrgn Ppp1r12a Hbb-b1  4 Itga3 Cd9 Vcp Il1b  5 Gsto1 Csf3r Klf9 Ppbp  6 Mmp2 Il1b Mprip Hba-a2  7 Mfge8 Gdpd3 Sdc4 kg:uc007pgs.1  8 Capg Ms4a1 Gpc5a kg:uc011yvj.1  9 Cd63 Hbb-b1 Vat1 Coro1a  10 Stub1 Beta-s Wdr92 Cd74  11 Lad1 kg:uc007pgs.1 S100a11 Gdpd3  12 Myo1h kg:uc011yvj.1 Clic4 Ccndbp1  13 Igfbp7 Rprl1 Dync1i2 kg:uc009cfw.1  14 Kcnn4 Pfn1 Nfkbiz kg:uc012enb.1  15 D8Ertd738e Clec1b Cyp2s1 Ptprc  16 Lamb3 Ptprc Esam Csf3r  17 Chi3l3 Stim1 Surf4 Rac2  18 Arl4c Ccndbp1 Krt19 Rprl1  19 Col18a1 Cap1 Bsg H2-Ab1  20 Atox1 Cd79b Tm4sf1 Epb4.1  21 Ly6a Alox12 Lgals3 Lyz2  22 Dmbt1 Hba-a2 Clic1 Ctla2b  23 Dync1h1 Ube2l6 Capns1 Pld4  24 Adipor2 Cat Igfbp6 kg:uc007pgt.1  25 Rpl37 Faim3 Rrbp1 Gng11  26 Kctd10 Dusp1 Calr Mepce  27 Col15a1 kg:uc007pgt.1 Rtf1 Tyrobp  28 Surf4 E2f2 Ildr2 Isca1  29 Dad1 Phospho1 Mark2 2810453l06Rik  30 Col4a1 Abi3 Mt1 Slc30a9  31 Ap2s1 Sorl1 Akr1b3 Treml2  32 Sdc1 Treml2 Gm6644 Srgn  33 Rpl35 Cytip Nkain4 Dcaf12  34 Sec61a1 B2m Ppp2ca Plek  35 Rras Fyb Akap2 Cat  36 Oraov1 Peli1 Hspb1 Alox12  37 Ndufa2 Plek Ptgis Fech  38 Anapc2 N4bp3 Msln Rbm5  39 Pitpna Fam117a Emp2 Cd97  40 Psap Srgn Capn2 March8  41 Atp5j2 Sept9 Rhoc Pnpo  42 Onecut2 kg:uc012hdk.1 Ptprf Phospho1  43 Hmga1 kg:uc009vev.1 Bcam Isg20  44 Pmepa1 Ptprcap Ogdh March2  45 S100a11 kg:uc007pgq.1 Sparc Lsp1  46 Rbp1 kg:uc007pgr.1 Ahnak 1810058l24Rik  47 Rpl36al kg:uc007vdl.1 Oasl2 Clec1b  48 S100a4 Ctla2b Wt1 Btg1  49 Atp6ap1 Myl9 Klf4 Laptm5  50 Ndufs2 Itpr2 Cdkn1a Nrgn  51 Anapc5 kg:uc012enb.1 Myl7 H2-Aa  52 Cox6b1 Isg20 Col1a2 Fyb  53 Krtcap2 Rasal3 Eif4a1 Cd24a  54 Atn1 Gng11 Rbpms Fnbp4  55 5730559C18Rik kg:uc009cfw.1 Emp3 Ehbp1l1  56 Pea15a Tmsb4x Scaf11 Ctla2a  57 Grcc10 Trem1 Col14a1 Sgk1  58 Lama5 Fech Ptrf Glyr1  59 Krt18 Epb4.1 Crip2 Myl9  60 Ccnd1 Sgk1 Ubxn4 Il2rg  61 Arhgef5 Dgkq Eif2s2 Mrps17  62 Golm1 Snap23 S100a6 Cdr2  63 Tff2 Usp25 Hectd1 Mkrn1  64 Plin2 Kif21b Zc3h15 Gart  65 H13 Irs2 Ube2d3 Lyz1  66 Rpl29 Pxk A130040M12Rik Vwf  67 1110034A24Rik Cyp4f18 Cd34 Gadd45a  68 Trim28 Map4k1 Igfbp5 Mpp1  69 Ltbp3 Isca1 C1s Stim1  70 Fkbp1a Itga4 Upk3b Psme3  71 Erp29 Dock2 Gpr133 Ets1  72 Muc1 Spib Dab2 Snap23  73 Lamc2 2810453l06Rik Serpinh1 Arhgdib  74 Plscr3 Cdr2 Upk1b Hmha1  75 Agrn Naa16 Sdf4 Itpr2  76 Park7 Arhgdib Ctbp2 Ubl7  77 Ctnnb1 Cd79a Psap Ddx58  78 Atp5g1 Rbm27 Arhgef12 Nfkbie  79 Eef1g Lmnb1 Copb2 Setd7  80 Nhp2 Slc25a37 Ctsl Stk24  81 Rrbp1 Klf6 Aldh1a2 Hvcn1  82 Sumo3 Hist1h1c Dcn Plekha2  83 Scyl1 Phip Timp3 Psme4  84 Cox6a1 Qrfp Xdh Ankrd44  85 Krt8 Fermt3 Irf7 B4galt5  86 Gsta4 Ptma Tmem151a Phf20  87 Ppp1r14b Etv3 Aebp1 Zc3hav1  88 Tnk1 Apobr C2 Rnf11  89 D19Wsu162e kg:uc008ewj.2 Spen Plk3  90 Ctsl Malat1 kg:uc007pfe.1 Fbxw5  91 Timp1 March8 Krt18 Emb  92 S100a6 Coro1a Arf4 kg:uc007vdl.1  93 Rps15 Rac2 Rab14 Taok2  94 Polr2j Glyr1 Tmem98 Dhrs11  95 Hspe1 Btg2 Prss23 Slc25a39  96 Lgals4 Mtf2 Egr1 Csk  97 Edf1 Nfkbie Perp Bcl2  98 Mtch1 Cd84 Csrp1 kg:uc009vev.1  99 Rnf187 AW549877 Pdpn Wipf1 100 Npy March2 Pdcd6ip Sept9 101 Cox5b Add3 Rpl37 Rnf10 102 Pak1 Ddx50 F11r Pml 103 Mmp7 Prkcb Gpm6a Cd9 104 Fxyd3 Klf2 Tuba1a D4Wsu53e 105 Cuta Dcaf12 Ctnna1 Traf7 106 Ndufb8 Il2rg Anxa8 Pitpnc1 107 Gps1 Selplg Tpm1 Mms19 108 Bud31 Cd37 S100a16 Naa16 109 Ppap2c Fastkd2 Chmp4b Sharpin 110 Dap Rsad2 Tbrg1 Capza1 111 Slc25a1 Msn C3 Rsad2 112 Chaf1a 2010321M09Rik Ptgs1 kg:uc012hdk.1 113 Asxl1 Kif2a Rhou Ghitm 114 Jmjd8 Cd97 Cdc42 Csnk1g1 115 Tecr Hvcn1 Gpx4 Dgkz 116 Mgp Nipsnap3b Ppib B2m 117 Uqcrh Uba7 Stub1 Irs2 118 Wdr38 1810058I24Rik Dmkn Emg1 119 Col4a2 Nfrkb Rnh1 Impact 120 Tnnt2 Pabpc1 Pdgfa Mylip 121 Ndufs8 Usp16 Rpl37a Psmb8 122 Tspan4 Pde1b Rabac1 Rfk 123 Agpat6 Ncoa4 Timp2 Map3k5 124 Timp3 Irf8 Serping1 Odc1 125 Ankrd50 Ppp1cb Rbm39 Slc11a2 126 Ube2d3 Rgs2 Tgoln1 Eif2b1 127 Sf1 Smyd4 Nfix kg:uc008wjd.1 128 Csnk1d Arid3b Brd4 Rexo1 129 Reg3b Sh3bgrl2 Tmem234 Ddx50 130 Flot2 Lyl1 Wbp5 Nipsnap3b 131 Lmna Prr13 Ppig Sp100 132 2310044H10Rik Plagl2 Cd63 Uggt1 133 H19 Nfkbia Col1a1 kg:uc007czl.1 134 Slc20a1 Eef1a1 Mt2 Arpc5 135 6720456B07Rik Brd2 Zbtb7c Nfrkb 136 Mdh2 Egr1 Npr1 Nap1l4 137 Eif6 Mkrn1 Tmem119 Fam117a 138 Phf5a Pld4 Atf3 Sipa1l1 139 Vps28 Aldh1a1 Ankhd1 Ttc1 140 Bag1 Dnajb9 Tmed10 kg:uc009vew.1 141 Cyc1 Gjb5 Slc6a4 142 Angptl4 Mtif2 Atxn7l3b 143 Lgals3 H2- Rpl29 DMb2 144 Farsb Sdpr Ccar1 145 Mbd3 4932438A13Rik Ltbp4 146 Timm13 Treml1 Scyl1 147 Tpd52l2 Nup153 Ap3d1 148 Ptprn Mpp1 Iqgap1 149 Crip2 Dhrs11 Cldn15 150 Raver1 Lrmp Spnb2 151 Eif2b2 Manf Ano1 152 Psma7 Mll3 Lrrn4 153 Rps6ka4 Fam116b Id3 154 Mgat4a B4galt5 Eif3a 155 Ifitm2 kg:uc009vew.1 Prkcdbp 156 Wars Ly6d Atp1a1 157 Capn5 Dguok Dnaja2 158 Bsg Pnpo Tubb4b 159 Sec16a Tmem175 Hnrnpab 160 Cldn7 Gm6548 Mmp14 161 Cox7a2 Rsrc2 Atp1b1 162 Nek6 Ccdc88b Psip1 163 Rpl39 Akna Mgll 164 Itpr3 Tsc22d3 Rnase4 165 Ctnna1 Txndc5 Ywhab 166 Tpd52 Tubb4a Clip1 167 Mlf2 Stx11 Syn3 168 Crip1 D4Wsu53e Myl12a 169 Fkbp4 Amfr Rbm25 170 Gprc5a Tti1 Arf2 171 Slc4a11 Fam175b Cav1 172 Syn3 Zfp36 Hnrnpc 173 Npc2 Ddx5 Syne2 174 Rpl32 Tlr7 Dst 175 Inf2 Rfk 176 Rps10 kg:uc007ded.1 177 Rps26 Gnb2 178 Rpl37a Tmed5 179 Ctxn1 Thbs1 180 Lrrc59 eg:320169:chr9:p 181 Dctn1 Zfp335 182 Mtap4 Emg1 183 Uqcr10 Trmt61a 184 Suds3 Adipor1 185 Ap1s1 Vwf 186 S100a1 Aatf 187 Atp5j Trib1 188 Aim1 Pcyt1a 189 Plec Stx18 190 Prom1 Trp53bp2 191 Rhoc Stk40 192 Mast3 Il18 193 Olfml3 1810014B01Rik 194 Uqcr11 Lcp2 195 Plp2 Gimap4 196 Spna2 Rabl2 197 1700017B05Rik Ncf2 198 Anxa4 eg:497210:chr14:m 199 Nudc Tpt1 200 Asl Mll5 201 Prkcsh H3f3a 202 Plod3 Tspan13 203 Ndufa9 Il10ra 204 Impdh2 Mdc1 205 Ccnl2 Stk24 206 Nedd8 Myst4 207 Atp6v1f Zdhhc20 208 Mt1 Eif2b1 209 Il4ra Exoc4 210 Cndp2 Wipf1 211 Aprt Impa1 212 Preb Tmem119 213 Ap3d1 Pml 214 Mcm6 Ubb 215 Ubr4 Zmat3 216 Pvrl2 Slc30a9 217 Snrpg Lat 218 Cycs Tgfb2 219 Efemp2 Ube2o 220 Cct4 Igfbp5 221 Gm20605 Tspan5 222 Smad3 Fmnl1 223 Card10 Fnbp4 224 Krt7 Extl3 225 Cct2 Adcy7 226 Coro1c Enpp4 227 Ltbr Sep15 228 Ric8 H2-Ab1 229 Ndufs6 Bnip3l 230 Fibp Slc11a2 231 Pold4 Stom 232 Rpl34 Mfhas1 233 Rpl34-ps1 Mettl1 234 Clic1 Rnf10 235 Eri3 kg:uc009cfd.1 236 Ets2 Klf4 237 Unc13a Psme4 238 Usmg5 Sema4a 239 Sh3pxd2b Ftl2 240 Wdr6 Atad1 241 Las1l Tspan31 242 Polr2f Srrm2 243 Vamp5 Rab5c 244 Endod1 Capza1 245 Snrpd2 H2-Aa 246 Tpi1 Fhl1 247 Wwp2 Cryab 248 Dalrd3 Arid4b 249 Iqgap1 Gart 250 Ahsa1 1110004F10Rik 251 Trim27 Rnf11 252 Serpinf1 Zc3hav1 253 D330041H03Rik kg:uc008btl.1 254 Ppp2r5d Rnf34 255 Minos1 Dmkn 256 Tsta3 Btg1 257 Prpsap1 Syt11 258 Sphk1 Mtdh 259 Ldha Med21 260 Abca3 Rnf2 261 B4galt3 Tcf12 262 Porcn Tacstd2 263 Tmc4 Madd 264 Serinc2 D16Ertd472e 265 Akr1b8 Pias1 266 Nudt4 Taok2 267 Atp5l Pold1 268 Psmc3 Cep110 269 Hint1 A930013F10Rik 270 Rpl41 Tcof1 271 Xpnpep1 kg:uc009bpd.1 272 Nav1 kg:u009bpr.2 273 Parva Capza2 274 Immt Ptp4a2 275 Pafah1b3 Fth1 276 Chid1 Mepce 277 Aldh1l1 Rexo1 278 Rpl31 Prg4 279 Wbp1 Ctla2a 280 Zfp622 Smarca5 281 2700060E02Rik Icam2 282 Hspa9 Pbx1 283 Tceb2 Gnl3l 284 Rpl36a Slc2a3 285 Pgs1 Nnmt 286 Mpnd Rb1cc1 287 Cdc42 Bpgm 288 Dhrs3 Lcp1 289 Hexa Sipa1l1 290 Cpsf1 Lilrb4 291 Mea1 Ankrd44 292 Polr2e Specc1 293 Ddb1 Rif1 294 Ptcd1 295 Atp5f1 296 Sec61b 297 Psmc5 298 Fam89b 299 Lama3 300 Tomm6 301 Mrpl28 302 Syngr2 303 Ngfrap1 304 Kcmf1 305 Tubb4b 306 Anapc11 307 Vcp 308 Arpp19 309 Pglyrp1 310 Rrp1 311 Gkn3 312 Atpif1 313 Prickle3 314 Map4k4 315 Arrdc1 316 C1qtnf6 317 Hras1 318 Lamb1 319 Eif3d 320 Snrpa 321 Tbrg1 322 Nxf1 323 Pdlim7 324 Add1 325 Pfdn5 326 Stk16 327 Gm17821 328 Csnk1e 329 Rrp7a 330 Psmb6 331 Snhg1 332 Ssr4 333 Ergic3 334 Rnaseh2a 335 kg:uc009cut.1 336 Bgn 337 Gm5506 338 Uqcrq 339 Tmem167 340 Nasp 341 Mif 342 Acaa2 343 Fam162a 344 Eif4ebp3 345 Nhp2l1 346 Prelid1 347 Gss 348 Lonp1 349 Srsf2 350 Igsf8 351 Ndufa7 352 Neat1 353 S100a13 354 Apoa1bp 355 Fam40a 356 Rps25 357 Eno1 358 Cldn2 359 Capn2 360 Glo1 361 Atp5c1 362 Rab2a 363 Rab25 364 Ncor2 365 Lgi4 366 Ier3 367 Tmem223 368 Slc9a3r2 369 Atp13a1 370 Rpn2 371 Acp5 372 Cct5 373 Sdf4 374 Mprip 375 Pmm2 376 Snx22 377 Arl2 378 1110008F13Rik 379 Polr1d 380 Dpm2 381 Cela1 382 2310016M24Rik 383 Cep250 384 Mybbp1a 385 Polr2g 386 Bag6 387 Cpxm1 388 Eif3m 389 Prr24 390 Sra1 391 Scara3 392 Reg1 393 Gas5 394 Hnrnpab 395 Mcpt2 396 Tgfbi 397 Capns1 398 Fdx1l 399 S100a16 400 Nap1l1 401 Swi5 402 Rpl38 403 Dctn2 404 Pdlim1 405 Gemin7 406 Pnpla6 407 Nono 408 Sla2 409 Idh3b 410 Ppp2r4 411 Map2k2 412 Ndufb10 413 Atp5d 414 Arfgap1 415 Tmbim1 416 Ergic1 417 Pdgfa 418 Ppp2ca 419 Hk1 420 Ltbp2 421 Trim35 422 Gtf2i 423 C1qb 424 Ankhd1 425 Podxl 426 Rps21 427 Huwe1 428 Pomp 429 Dpp3 430 Fkbp8 431 Itga5 432 Hes6 433 Mrpl11 434 Poldip3 435 Scd2 436 Tmem55b 437 Ndufa13 438 Dcakd 439 Ubqln1 440 Gpx4 441 Cyb561 442 Gmppa 443 Ncaph2 444 Pdha1 445 Ndufs4 446 Fcer1g 447 Myof 448 Ppib 449 Mrpl52 450 Tes 451 Emp3 452 Ndufa11 453 Tor1aip2 454 Anp32b 455 Tnk2 456 Mcpt1 457 Ssr2 458 Psmb3 459 2700081O15Rik 460 Pcbd1 461 Eif1ax 462 Pmm1 463 Ptprk 464 Hadha 465 Calu 466 Fam73a 467 Atp5e 468 Hsd17b10 469 Rbm39 470 Egfl7 471 Psmc1 472 Perp 473 Lman2 474 Galnt1 475 Rbx1 476 Lemd2 477 Zglp1 478 Ing4 479 kg:uc008oow.1 480 1500012F01Rik 481 Cox4i1 482 kg:uc008bcq.1 483 Ubap2l 484 Pafah1b2 485 Mrpl13 486 Nucb1 487 Fbn1 488 Adrm1 489 Itgb4 490 Ctss 491 Plbd2 492 Ptpmt1 493 Sap30l 494 Ppp1r12c 495 Sgta 496 Acrbp 497 Higd2a 498 Higd1a 499 Tmem208 500 Cdh1 501 Ube2d2a 502 Suv39h1 503 Rabac1 504 Anxa5 505 Ubxn6 506 Tpm1 507 Hmga2 508 Cnbp 509 Rpl21 510 Ndufb5 511 Sec31a 512 Znhit1 513 Cyb5b 514 Sfn 515 Ccdc12 516 Elovl1 517 Psmb5 518 Slc25a11 519 Psmd2 520 Nsun2 521 Slc50a1 522 Eme1 523 Bnip2 524 Pxdn 525 Mad2l2 526 Pdcd6 527 2010107E04Rik 528 Abhd11 529 Carkd 530 Polr2l 531 Ppdpf 532 Cib1 533 Dgcr2 534 Timm50 535 Mrps24 536 Abhd12 537 Brf1 538 Man1b1 539 kg:uc012cgd.1 540 Gpaa1 541 Fmnl3 542 Mapk3 543 C1qc 544 Pgls 545 Cp 546 Serh1 547 2610203C20Rik 548 Hsbp1 549 Tmem214 550 Akt1 551 kg:uc007pfe.1 552 Tmed10 553 Ttll3 554 2200002D01Rik 555 Tnfrsf23 556 Sgsm3 557 Atp9a 558 Lcn2 559 Pdrg1 560 Tspan9 561 Nrd1 562 Rin1 563 Ndufv1 564 Naa10 565 Wnk1 566 Heatr7a 567 Slc4a2 568 Ggct 569 5730403B10Rik 570 Sh3glb2 571 Pfkl 572 Tspan3 573 Gns 574 Sdcbp2 575 C130074G19Rik 576 Cotl1 577 Tubb5 578 Sec11c 579 Pigq 580 Zc3h15 581 Lsmd1 582 Ppa1 583 Chmp4b 584 Sepn1 585 Angptl2 586 Itpripl2 587 Ddx1 588 Hbxip 589 Cdk2ap1 590 Clta 591 Cpsf3l 592 Apoe 593 Ift46 594 Sae1 595 Gpi1 596 Gorasp2 597 1500032L24Rik 598 Nsmce4a 599 Dlst 600 Bap1 601 Pitpnb 602 Meg3 603 Cyth2 604 Atp5o 605 Gon4l 606 Sox11 607 Cxxc5 608 Avil 609 Alcam 610 Eif3f 611 Cygb 612 Eif1ad 613 Polr3h 614 Araf 615 Gkn1 616 Rhog 617 Mtap 618 Eif4ebp1 619 Akr1a1 620 Trip6 621 Prdx6 622 2410015M20Rik 623 Rps6 624 Rps23 625 Stxbp2 626 Rps19 627 Ykt6 628 Atp5g2 629 Serpinb1a 630 Col7a1 631 Mrps6 632 Lgals9 633 Rcn3 634 Trim44 635 Surf2 636 Rps29 637 Cdipt 638 Lmf2 639 Psenen 640 Ltf 641 Mpzl1 642 Psmd6 643 Cttn 644 Tmc6 645 2500003M10Rik 646 Atp6v0a1 647 Med8 648 Prrx2 649 Atp5b 650 Smurf1 651 Carhsp1 652 Tpcn1 653 Ndufb9 654 Pih1d1 655 Hnrnpa0 656 Fn1 657 2810428I15Rik 658 0610012G03Rik 659 Ube2i 660 Anxa3 661 Msto1 662 Eng 663 0910001L09Rik 664 Rpl10 665 kg:uc007xxx.1 666 Mosc2 667 Vps37c 668 Sgpl1 669 Fam166a 670 Polr2b 671 Fam101b 672 Nupr1 673 Lsm4 674 Rpl36 675 0610007C21Rik 676 Psmc2 677 Supt6h 678 Rps13 679 5430437P03Rik 680 Dsp 681 Ddx56 682 Tsc2 683 Trmt2a 684 Vdac2 685 Cant1 686 Eif4h 687 Puf60 688 A430105I19Rik 689 Cacnb3 690 Prdx4 691 March5 692 Ccar1 693 Npepl1 694 Fermt1 695 Use1 696 Axl 697 Slc39a4 698 1110008P14Rik 699 Sema4g 700 Timm8b 701 Krt23 702 Rpl28 703 Lgals3bp 704 Hdgf 705 1110005A03Rik 706 Impdh1 707 Mtmr11 708 Msln 709 Zdhhc3 710 Znrf1 711 Aldh16a1 712 Bloc1s1 713 Prkag1 714 Plxnb1 715 Crat 716 Phpt1 717 5930434B04Rik 718 Kpnb1 719 Nme2 720 E430025E21Rik 721 Smyd2 722 Cyhr1 723 Mvp 724 Rps27l 725 Rbp4 726 Cars 727 kg:uc012ctw.1 728 Ssr1 729 Ssu72 730 Usp48 731 Atp5k 732 Lrrk1 733 BC056474 734 Epn1 735 Trappc1 736 Clk2 737 Sugt1 738 Nenf 739 kg:uc009cuu.1 740 Ubap2 741 Rps20 742 Atp5h 743 9430008C03Rik 744 Kars 745 Mrpl37 746 Aimp1 747 Trmt1 748 Hspa4 749 Cd164 750 9430023L20Rik 751 Rnf4 752 H1f0 753 C1qtnf1 754 Srd5a1 755 1500010J02Rik 756 Rpl35a 757 Cand2 758 C630004H02Rik 759 Acsbg1 760 Derl1 761 Cbx5 762 Tmem63a 763 Hgfac 764 Stx5a 765 Bri3 766 Tomm20 767 Fam20c 768 Cox6c 769 Tm2d2 770 Plekhb2 771 Ramp1 772 2410001C21Rik 773 Tardbp 774 Pebp1 775 kg:uc008gbp.1 776 Eif3b 777 Ccna2 778 Ptges 779 kg:uc007hyr.2 780 Wbp5 781 Chchd2 782 Fdft1 783 Srm 784 Gtf3a 785 D0H4S114 786 1810009A15Rik 787 Rps27 788 Tmem176b 789 Ndufc1 790 Lasp1 791 Fam108a 792 Mapk8ip3 793 Copa 794 Serpina3n 795 Rps17 796 Dnpep 797 Lbp 798 Krt19 799 Ei24 800 Ap1b1 801 Mogs 802 Uba1 803 Postn 804 Phf23 805 Paox 806 Nploc4 807 Ndufv2 808 Actr1a 809 Mxd3 810 Pfdn1 811 Ide 812 Foxp4 813 1810013D10Rik 814 2310007B03Rik 815 Xab2 816 Agr2 817 Dctn3 818 Urm1 819 H2-Ke2 820 Spint1 821 Slc38a2 822 Ube2z 823 Ctrb1 824 Fam195b 825 Suclg1 826 Ube2l3 827 Rpn1 828 Mrps7 829 Tsg101 830 Drosha 831 Arfip2 832 Mrto4 833 Grlf1 834 Sort1 835 Oaf 836 Ints1 837 Slc44a2 838 Dph3 839 Gramd1a 840 Fkbp9 841 Fam149a 842 1810035L17Rik 843 kg:uc007fte.1 844 Eif2s1 845 Smpd1 846 Eef1b2 847 Actr10 848 Rab11fip5 849 Ypel3 850 Flnb 851 Tcn2 852 Crlf1 853 Map3k15 854 Cul7 855 Atp6v1g1 856 Ncbp1 857 Atp1b3 858 Mtif3 859 Aldoa 860 Htra1 861 Rab14 862 Ppm1a 863 Ndufb11 864 Kansl3 865 Rab24 866 Bcl2l1 867 Lgals1 868 Samm50 869 Mrps33 870 Anxa1 871 Chchd1 872 Mapre1 873 Ctbp2 874 Rnps1 875 Spg7 876 Tnfrsf12a 877 H6pd 878 Myo7a 879 Mcm7 880 Psmd13 881 Mrpl54 882 Atp6v0b 883 Prdx1 884 Elof1 885 Rexo4 886 Mrps18a 887 Dpcd 888 D2Wsu81e 889 Cd99l2 890 Synpo 891 Atp2a2 892 Cdc5l 893 Stard7 894 Atp13a2 895 Sdha 896 Hdac6 897 Krt20 898 Ppp6r3 899 1700037H04Rik 900 Napa 901 PgP 902 Cnih 903 Atg4b 904 Cox8a 905 Srp68 906 St13 907 Gng12 908 Cfdp1 909 Rcc2 910 Pisd- ps1 911 Ivns1abp 912 Mpv17l2 913 Ssna1 914 Gnl1 915 Tmem111 916 Hbs1l 917 Agpat3 918 Col6a2 919 March6 920 Usp39 921 Rps11 922 Ahnak 923 Lcmt1 924 Ddx41 925 H2afv 926 Fau 927 Tuba1c

The gene names listed in Table 13 and 12 are common names. NCBI Gene ID numbers for each of the genes listed in Table 13 and 12 can be obtained by searching the “Gene” Database of the NCBI (available on the World Wide Web at http://www.ncbi.nlm.nih.gov/) using the common name as the query and selecting the first returned Homo sapiens gene. Other genes may be obtained using the UCSC genome browser (available on the World Wide Web at http://genome.ucsc.edu) using the Gene Sorter function. In certain embodiments, the marker gene(s) are selected from the genes listed in Table 13 and/or 12.

In some embodiments, the marker gene(s) is selected from a marker gene indicated to be upregulated in at least one type of CTC in Table 13, e.g. marker genes 1-142. In some embodiments, the marker gene(s) is selected from a marker gene indicated to be upregulated in at least one type of CTC in Table 12, e.g. marker genes listed in the columns labeled “CTC-c vs. Primary Tumor Enriched Gene” or “CTC-c vs. WBC”.

In a CTC, the marker genes listed in Table 13 or 12 can be upregulated, e.g. for marker genes listed in Table 13 and/or 12, if the measured marker gene expression in a cell or sample is higher as compared to a reference level of that marker gene's expression, then the cell is identified as a CTC and/or the sample is identified as comprising CTCs. Preferably, once looks at a statistically significant change. However, even if a few genes in a group do not differ from normal, a sample can be identified as comprising CTCs if the overall change of the group shows a significant change, preferably a statistically significant change. All possible combinations of 2 or more of the indicated markers are contemplated herein. 

1. A method of detecting circulating tumor cells (CTCs) in a sample, the method comprising: measuring the level of a PC-CTC marker gene expression product in the sample; wherein a level of the marker gene expression product which is greater than a reference level indicates the presence of CTCs in the sample.
 2. The method of claim 1, wherein the CTCs are pancreatic cancer CTCs.
 3. The method of claim 1, wherein the method further comprises a first step of isolating the CTCs from the sample.
 4. The method of claim 1, wherein the expression product is a nucleic acid or a polypeptide. 5.-7. (canceled)
 8. The method of claim 1, wherein the CTC marker gene is selected from Table 7; Table 8; or Table
 14. 9. The method of claim 1, wherein the CTC marker gene is selected from the group consisting of: ABI3BP; ADAMTS5; ADAMTSL1; ANG; ARSA; C1RL; C3; C4A; C4B; CCDC80; CD109; CHI3L1; CLEC3B; CMTM3; CMTM7; COL14A1; COL1A2; COL3A1; COL4A6; CSF1; DAG1; DCN; DMKN; FBLN1; FGF1; FMOD; GPC3; GPC4; HMGB1; IFNAR2; IGFBP5; IL16; LAMA4; LTBP4; MFAP1A; NID2; OGN; PDAP1; PF4; PLAT; PODN; PRELP; RSPO1; SERPING1; SLURP1; SOD3; SPARC; SPOCK2; SPON2; SULF1; SULF2; TGFB2; TGM2; THBD; THBS1; THSD4; TIMP2; TNXB; TPT1; TWSG1 and WNT4.
 10. The method of claim 1, wherein the CTC marker gene is selected from the group consisting of: ALDH1A1; ALDH1A2; IGFBP5; KLF4; DCN; SPARC; WNT; TGFB2; VEGF; COL1A2; COL3A1; and TIMP2.
 11. The method of claim 1, wherein the CTC marker gene is selected from the group consisting of: ALDH1A2; IGFBP5; KLF4; DCN; and SPARC.
 12. The method of claim 1, wherein the CTC marker gene is selected from the group consisting of: ALDH1A2; IGFBP5; KLF4; and DCN.
 13. The method of claim 1, wherein the CTC marker gene is selected from the group consisting of: TPT1; HMGB1; SPON 2; SPARC; and ARSA.
 14. The method of claim 1, wherein the CTC marker gene is selected from the group consisting of: IL6ST; ARSA; TIMP2; CD55; SULF2; ITGA6; SDC4; CDON; and SV2A.
 15. A method of treating cancer in a subject, the method comprising administering a therapeutically effective amount of a CTC marker gene-targeted therapy to the subject.
 16. The method of claim 15, wherein the cancer is pancreatic cancer.
 17. The method of claim 15, wherein the CTC marker gene-targeted therapy comprises an inhibitor of a CTC marker gene.
 18. The method of claim 17, wherein the inhibitor is an antibody reagent.
 19. The method of claim 17, wherein the inhibitor is an inhibitory nucleic acid reagent.
 20. The method of claim 15, wherein the CTC marker gene-targeted therapy comprises a CTC marker gene-binding antibody reagent and a chemotherapeutic agent.
 21. The method of claim 15, wherein the subject is a subject determined to have an elevated level of CTCs and/or an elevated level of a CTC marker gene present in the blood and/or stroma of the cancer.
 22. The method of claim 15, wherein the CTC marker gene-targeted therapy comprises a CTC marker gene-binding antibody reagent that binds a marker gene selected from the group consisting of: IL6ST, SULF2, and SV2A. 23.-51. (canceled) 